POLYNUCLEOTIDES ENCODING METHYLMALONYL-CoA MUTASE

ABSTRACT

The disclosure relates to polynucleotides comprising an open reading frame of linked nucleosides encoding human methylmalonyl-CoA mutase precursor, human methylmalonyl-CoA mutase (MCM) mature form, or functional fragments thereof. In some embodiments, the disclosure includes methods of treating methylmalonic acidemia in a subject in need thereof comprising administering an mRNA encoding an MCM polypeptide.

BACKGROUND OF THE DISCLOSURE

Methylmalonic acidemia (MMA) is a metabolic disorder characterized bythe abnormal buildup of the metabolic byproduct methylmalonic acid inpatients. MMA causes developmental delay, intellectual disability,kidney disease, coma, or even death. MMA is also referred to asmethylmalonic aciduria. It has an estimated incidence of 1 in 50,000 to100,000. Current treatment for MMA is primarily via dietary control tolimit the usage of metabolic pathways that lead to methylmalonic acidformation. In serious cases, kidney and liver transplants have also beenperformed to provide a new reservoir of cells that can properlymetabolize and remove the methylmalonic acid. However, none of thesetreatments completely or reliably controls the disorder. As such thereis a need for improved therapy to treat MMA.

The principal gene associated with MMA is methylmalonyl-CoA mutase(NM_000255; NP_000246; also referred to as MCM or MUT). MCM is ametabolic enzyme (E.C. 5.4.99.2) that plays a critical role in thecatabolism of various amino acids, fatty acids, and cholesterol. MCM'sbiological function is to isomerize L-methylmalonyl-CoA intosuccinyl-CoA, a Krebs cycle intermediate. MCM localizes to themitochondria of cells, exists as a homodimer in its native form and isadenosylcobalamin-dependent. The precursor form of human MCM is 750amino acids, while its mature form is 718 amino acids—a 32 amino acidleader sequence is cleaved off by mitochondrial importation andprocessing machinery. This leader sequence is variously referred to asMCM's mitochondrial targeting peptide, mitochondrial targeting sequence,or mitochondrial transit peptide.

A complete or partial loss of MCM function leads to buildup of abnormalmetabolites and metabolic intermediates upstream of MCM, such asmethylmalonic acid, propionyl-carnitine, acetyl-carnitine,propionyl-CoA, D-methylmalonyl-CoA and L-methylmalonyl-CoA. For example,loss of MCM has been reported to lead to a 1000-fold increase in themethylmalonic acid. Nonetheless, there is no currently availabletherapeutic to treat MMA.

SUMMARY OF THE DISCLOSURE

The present disclosure provides methods of treating methylmalonicacidemia in a subject, the methods comprising administering to thesubject an effective amount of a polynucleotide comprising an mRNAencoding an MCM polypeptide, wherein the administration alleviates thesymptoms of methylmalonic acidemia in the subject. The presentdisclosure also provides compositions comprising a polynucleotidesequence encoding an MCM polypeptide. In some embodiments, thecompositions include a delivery agent.

In some embodiments, the composition comprises a polynucleotide thatcomprises an open reading frame (ORF) encoding an MCM polypeptide and adelivery agent, wherein the delivery agent comprises a compound havingthe formula (I)

or a salt or stereoisomer thereof, wherein

R₁ is selected from the group consisting of C₅₋₂₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR,

—CHQR, —CQ(R)₂, and unsubstituted C₁₋₆ alkyl, where Q is selected from acarbocycle, heterocycle,

—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—N(R)₂, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂,—N(R)C(S)N(R)₂, and —C(R)N(R)₂C(O)OR, and each n is independentlyselected from 1, 2, 3, 4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,

—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; and

provided when R₄ is —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, or —CQ(R)₂, then(i) Q is not —N(R)₂ when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or7-membered heterocycloalkyl when n is 1 or 2.

In some embodiments, the delivery agent further comprises aphospholipid, a structural lipid, a PEG lipid, or any combinationthereof.

In some embodiments, the polynucleotides comprise an ORF havingsignificant sequence similarity to a polynucleotide selected from thegroup of SEQ ID NOs: 1-207, 732-765, and 772, wherein the ORF encodes anMCM polypeptide. In some embodiments, the polynucleotides comprise anORF having significant sequence similarity to a polynucleotide selectedfrom the group of SEQ ID NOs: 151, 152, 153, 154, 732, 733, and 734(FIGS. 9-15), wherein the ORF encodes an MCM polypeptide. In someembodiments, the polynucleotide comprises an ORF having significantsequence similarity to SEQ ID NO: 734 (FIG. 11), wherein the ORF encodesan MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 99% or 100% sequence identity tonucleotide 97 to nucleotide 2250 of SEQ ID NO: 734, wherein the ORFencodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF sequence having at least 98%, at least 99%, or 100%sequence identity to nucleotide 97 to nucleotide 2250 of SEQ ID NO: 732,wherein the ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to a sequence selected from the group consistingof nucleotides 97 to nucleotides 2250 of SEQ ID NOs: 182, 733, and 741,wherein the ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to a sequence selected from thegroup consisting of nucleotides 97 to nucleotides 2250 of SEQ ID NOs:735, 736, 738, 743, 744, 748, 749, 750, 754, 755, 758, 762, and 765,wherein the ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF sequence having at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% sequence identity to a sequenceselected from the group consisting of nucleotides 97 to nucleotides 2250of SEQ ID NOs: 180, 187, 737, 739, 740, 742, 745, 746, 747, 751, 752,753, 757, 759, 760, 761, 763, and 764, wherein the ORF encodes an MCMpolypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to asequence selected from the group consisting of nucleotides 97 tonucleotides 2250 of SEQ ID NO: 181 and 756, wherein the ORF encodes anMCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to a sequence selected from the group consisting of nucleotides97 to nucleotides 2250 of SEQ ID NO: 154, 165, 171, 173, and 175,wherein the ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to a sequence selected from the group consistingof nucleotides 97 to nucleotides 2250 of SEQ ID NO: 151, 152, 153, 163,164, 166, 167, 168, 169, 170, 172, 177, 178, 179, 195, and 204, whereinthe ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to a sequence selected from thegroup consisting of nucleotides 97 to nucleotides 2250 of SEQ ID NOs:156, 157, 158, 159, 160, 161, 162, 174 and 176, wherein the ORF encodesan MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to a sequenceselected from the group consisting of nucleotides 97 to nucleotides 2250of SEQ ID NOs: 155 and 203, wherein the ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to a sequence selected from the group consistingof nucleotides 97 to nucleotides 2250 of SEQ ID NOs: 64, 66, 71, 91, and128, wherein the ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to a sequence selected from thegroup consisting of nucleotides 97 to nucleotides 2250 of SEQ ID NOs: 9,11, 18, 19, 21, 22, 23, 24, 32, 33, 37, 39, 40, 44, 45, 47, 50, 51, 52,55, 57, 61, 65, 70, 79, 84, 86, 88, 90, 92, 98, 100, 115, 117, 126, 129,135, 136, 137, 144, 148, 150, 184, 190, 191, and 206, wherein the ORFencodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to a sequenceselected from the group consisting of nucleotides 97 to nucleotides 2250of SEQ ID NOs: 3, 5, 6, 8, 10, 12, 14, 16, 17, 20, 27, 28, 29, 31, 34,35, 36, 38, 41, 42, 43, 46, 48, 49, 53, 54, 56, 58, 60, 63, 68, 69, 74,77, 78, 80, 83, 85, 87, 93, 95, 96, 97, 99, 102, 103, 104, 105, 107,110, 112, 113, 114, 116, 119, 120, 122, 123, 124, 125, 127, 131, 132,133, 134, 138, 139, 140, 141, 142, 143, 147, 149, 183, 186, 188, 189,192, 193, 194, 196, 197, 198, 199, 200, 201, 202, 205, and 207, whereinthe ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 79%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to asequence selected from the group consisting of nucleotides 97 tonucleotides 2250 of SEQ ID NOs: 1, 2, 4, 7, 13, 15, 25, 26, 30, 59, 62,67, 72, 73, 75, 76, 81, 82, 89, 94, 101, 106, 108, 109, 111, 118, 121,130, 145, 146, and 185, wherein the ORF encodes an MCM polypeptide.

In some embodiments, the polynucleotides further comprise a nucleotidesequence encoding a transit peptide, e.g., mitochondrial transitpeptide. The mitochondrial transit peptide can be any peptide thatfacilitates the transport of MCM to mitochondria or localization of MCMin mitochondria. In some embodiments, the polynucleotide comprises anucleotide sequence encoding a mitochondrial transit peptide selectedfrom the group listed in Table 1 (SEQ ID NOs: 251 to 265). In someembodiments, the polynucleotide comprises a nucleotide sequence encodinga mitochondrial transit peptide selected from the group consisting ofSEQ ID NOs: 270 to 719.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 99% or 100% sequence identity tonucleotide 1 to nucleotide 2250 of SEQ ID NO: 734, wherein the ORFencodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 98%, at least 99%, or 100% sequenceidentity to nucleotide 1 to nucleotide 2250 of SEQ ID NO: 732, whereinthe ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to a sequence selected from the group consistingof nucleotides 1 to nucleotides 2250 of SEQ ID NO: 182 and 733, whereinthe ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to a sequence selected from thegroup consisting of nucleotides 1 to nucleotides 2250 of SEQ ID NOs:735, 741, 743, 744, 748, 758, 762, and 765, wherein the ORF encodes anMCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to a sequenceselected from the group consisting of nucleotides 1 to nucleotides 2250of SEQ ID NOs: 180, 181, 736, 738, 739, 740, 742, 746, 747, 749, 750,751, 752, 753, 754, 755, 757, 759, 760, 761, and 763, wherein the ORFencodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to asequence selected from the group consisting of nucleotides 1 tonucleotides 2250 of SEQ ID NO: 745, 756, and 764, wherein the ORFencodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to a sequence selected from the group consisting of nucleotides1 to nucleotides 2250 of SEQ ID NO: 154, 165, 171, 173, and 175, whereinthe ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to a sequence selected from the group consistingof nucleotides 1 to nucleotides 2250 of SEQ ID NO: 151, 152, 153, 163,166, 167, 168, 169, 170, 172, 177, 178, 179, 187, and 204, wherein theORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to a sequence selected from thegroup consisting of nucleotides 1 to nucleotides 2250 of SEQ ID NO: 156,157, 158, 159, 160, 162, 164, 174, 176, 195, and 737, wherein the ORFencodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to a sequenceselected from the group consisting of nucleotides 1 to nucleotides 2250of SEQ ID NOs: 155, 161, and 203, wherein the ORF encodes an MCMpolypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to a sequence selected from the group consistingof nucleotides 1 to nucleotides 2250 of SEQ ID NOs: 71 and 128, whereinthe ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to a sequence selected from thegroup consisting of nucleotides 1 to nucleotides 2250 of SEQ ID NOs: 4,6, 8, 9, 11, 19, 22, 23, 24, 32, 33, 37, 40, 44, 45, 47, 51, 61, 64, 65,66, 79, 84, 86, 90, 91, 92, 100, 101, 112, 115, 117, 126, 129, 135, 136,146, 148, 184, 190, and 191, wherein the ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to a sequenceselected from the group consisting of nucleotides 1 to nucleotides 2250of SEQ ID NOs: 2, 3, 5, 7, 10, 12, 13, 14, 15, 16, 18, 20, 21, 26, 27,28, 29, 31, 34, 36, 38, 39, 41, 42, 43, 46, 48, 49, 52, 53, 54, 55, 56,57, 58, 59, 60, 62, 68, 69, 70, 72, 73, 74, 76, 77, 80, 83, 85, 88, 95,96, 97, 98, 102, 104, 105, 106, 107, 108, 109, 110, 113, 114, 120, 121,122, 123, 124, 127, 131, 132, 133, 134, 137, 138, 139, 140, 141, 142,144, 145, 147, 149, 150, 186, 188, 189, 192, 193, 194, 196, 198, 199,200, 202, 205, 206, and 207, wherein the ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 79%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to asequence selected from the group consisting of nucleotides 1 tonucleotides 2250 of SEQ ID NOs: 1, 17, 25, 30, 35, 50, 63, 67, 75, 78,81, 82, 87, 89, 93, 94, 99, 103, 111, 116, 118, 119, 125, 130, 143, 183,185, 197, and 201, wherein the ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to polynucleotides thatencode functional MCMs or fragments thereof. In some embodiments, thedisclosure provides polynucleotides that encode functional human MCMs(SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ IDNO: 212, and SEQ ID NO: 213). In some embodiments, the disclosureprovides polynucleotides that encode functional MCM polypeptides havingat least one point mutation in the MCM sequence, while still retainingMCM enzymatic activity. In some embodiments, the encoded MCM polypeptidecomprises one or more of the point mutations V69, T499, H532, A598, andV671, as defined by the polypeptide sequences in SEQ ID NO: 209, SEQ IDNO: 210, SEQ ID NO: 211, SEQ ID NO: 212, and SEQ ID NO: 213,respectively. In some embodiments, the polynucleotides are fully orpartially modified (e.g., chemically and/or structurally) in a manner asto avoid the deficiencies of other molecules of the art. Thepolynucleotides of the disclosure can be synthesized as an IVTpolynucleotide, chimeric polynucleotide or a circular polynucleotide andsuch embodiments are contemplated.

In some embodiments, the polynucleotide is a DNA or RNA that comprisesat least one chemically modified nucleoside. In some embodiments, the atleast one chemically modified nucleoside is selected from any of thosedescribed herein.

In some embodiments, the polynucleotide further comprises or encodes a5′ UTR. In other embodiments, the polynucleotide further comprises orencodes a 3′ UTR. In some embodiments, the UTR comprises or encodes amiRNA (e.g., miR-142-3p, miR-142-5p, miR-126-3p, and/or miR-126-5p). Insome embodiments, the polynucleotide further comprises a 5′ terminalcap. In some embodiments, the polynucleotide further comprises orencodes a 3′ polyA tail.

In some embodiments, the polynucleotide is RNA, e.g., mRNA. In someembodiments, the mRNA comprises the sequences listed in SEQ ID NOs:766-771.

In some embodiments, the polynucleotide is an RNA polynucleotide that isformulated in a lipid nanoparticle (LNP) carrier.

The disclosure is also directed to a method of treating methylmalonicacidemia in a subject, the method comprising administering to thesubject an effective amount of a polynucleotide comprising an mRNAencoding an MCM polypeptide, wherein the administration alleviates thesymptoms of methylmalonic acidemia in the subject. In some embodiments,the polynucleotide useful for the disclosure is any one of thepolynucleotides encoding an MCM polypeptide described herein or isformulated as any one of the compositions described herein.

In some embodiments, the disclosure includes a method of reducing thelevel of a metabolite associated with methylmalonic acidemia in asubject in need thereof, the method comprising administering to thesubject an effective amount of a polynucleotide comprising an mRNAencoding an MCM polypeptide. In some embodiments, the polynucleotide isa polynucleotide described elsewhere herein, or is formulated as acomposition described herein. In certain embodiments, the polynucleotidereduces the level of methylmalonic acid present in the subject after theadministration by at least about 10%, at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, or about 100%.In other embodiments, after the administration, the polynucleotidereduces the level of propionyl-carnitine present in the subject by atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, or about 100%. In yet otherembodiments, the polynucleotide reduces the level of acetyl-carnitinepresent in the subject after the administration by at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, or about 100%. In certain embodiments, one or moremetabolites associated with methylmalonic acidemia are reduced after theadministration within one day, within two days, within three days,within four days, within five days, within seven days, within one week,within two weeks, within three weeks, or within one month of theadministration of the polynucleotide.

The details of various embodiments of the disclosure are set forth inthe description below. Other features, objects, and advantages of thedisclosure will be apparent from the description and the drawings, andfrom the claims.

EMBODIMENTS

E1. A composition comprising a polynucleotide that comprises an openreading frame (ORF) encoding an MCM polypeptide and a delivery agent,wherein the delivery agent comprises a compound having the formula (I)

or a salt or stereoisomer thereof, wherein

R₁ is selected from the group consisting of C₅₋₂₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR,

—CHQR, —CQ(R)₂, and unsubstituted C₁₋₆ alkyl, where Q is selected from acarbocycle, heterocycle,

—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—N(R)₂, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂,—N(R)C(S)N(R)₂, and —C(R)N(R)₂C(O)OR, and each n is independentlyselected from 1, 2, 3, 4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,

—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; and

provided when R₄ is —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, or —CQ(R)₂, then(i) Q is not —N(R)₂ when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or7-membered heterocycloalkyl when n is 1 or 2.

E2. The composition of embodiment 1, wherein the ORF has at least 80%,at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to a sequence selected from the group consistingof nucleotides 97 to nucleotides 2250 of SEQ ID NOs: 1 to 207, 732 to765, and 772.

E3. The composition of embodiment 1 or 2, wherein the ORF has

(i) at least 99% or 100% sequence identity to nucleotide 97 tonucleotide 2250 of SEQ ID NO: 734,

(ii) at least 98%, at least 99%, or 100% sequence identity to nucleotide97 to nucleotide 2250 of SEQ ID NO: 732,

(iii) at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identityto a sequence selected from the group consisting of nucleotides 97 tonucleotides 2250 of SEQ ID NOs: 182, 733, and 741;

(iv) at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a sequence selected from the group consisting ofnucleotides 97 to nucleotides 2250 of SEQ ID NOs: 735, 736, 738, 743,744, 748, 749, 750, 754, 755, 758, 762, and 765;

(v) at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% sequence identity to a sequence selected from the groupconsisting of nucleotides 97 to nucleotides 2250 of SEQ ID NOs: 180,187, 737, 739, 740, 742, 745, 746, 747, 751, 752, 753, 757, 759, 760,761, 763, and 764;

(vi) at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to a sequence selected fromthe group consisting of nucleotides 97 to nucleotides 2250 of SEQ IDNOs: 181 and 756;

(vii) at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% sequence identity to a sequenceselected from the group consisting of nucleotides 97 to nucleotides 2250of SEQ ID NO: 154, 165, 171, 173, and 175;

(viii) at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identityto a sequence selected from the group consisting of nucleotides 97 tonucleotides 2250 of SEQ ID NO: 151, 152, 153, 163, 164, 166, 167, 168,169, 170, 172, 177, 178, 179, 195, and 204;

(ix) at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a sequence selected from the group consisting ofnucleotides 97 to nucleotides 2250 of SEQ ID NOs: 156, 157, 158, 159,160, 161, 162, 174 and 176;

(x) at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% sequence identity to a sequence selected from the groupconsisting of nucleotides 97 to nucleotides 2250 of SEQ ID NOs: 155 and203;

(xi) at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identityto a sequence selected from the group consisting of nucleotides 97 tonucleotides 2250 of SEQ ID NOs: 64, 66, 71, 91, and 128;

(xii) at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a sequence selected from the group consisting ofnucleotides 97 to nucleotides 2250 of SEQ ID NOs: 9, 11, 18, 19, 21, 22,23, 24, 32, 33, 37, 39, 40, 44, 45, 47, 50, 51, 52, 55, 57, 61, 65, 70,79, 84, 86, 88, 90, 92, 98, 100, 115, 117, 126, 129, 135, 136, 137, 144,148, 150, 184, 190, 191, and 206;

(xiii) at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% sequence identity to a sequence selected from the groupconsisting of nucleotides 97 to nucleotides 2250 of SEQ ID NOs: 3, 5, 6,8, 10, 12, 14, 16, 17, 20, 27, 28, 29, 31, 34, 35, 36, 38, 41, 42, 43,46, 48, 49, 53, 54, 56, 58, 60, 63, 68, 69, 74, 77, 78, 80, 83, 85, 87,93, 95, 96, 97, 99, 102, 103, 104, 105, 107, 110, 112, 113, 114, 116,119, 120, 122, 123, 124, 125, 127, 131, 132, 133, 134, 138, 139, 140,141, 142, 143, 147, 149, 183, 186, 188, 189, 192, 193, 194, 196, 197,198, 199, 200, 201, 202, 205, and 207; or

(xiv) at least 79%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to a sequence selected fromthe group consisting of nucleotides 97 to nucleotides 2250 of SEQ IDNOs: 1, 2, 4, 7, 13, 15, 25, 26, 30, 59, 62, 67, 72, 73, 75, 76, 81, 82,89, 94, 101, 106, 108, 109, 111, 118, 121, 130, 145, 146, and 185.

E4. The composition of any one of embodiments 1 to 3, wherein the ORFfurther comprises a nucleic acid sequence encoding a transit peptide.

E5. The composition of embodiment 4, wherein the transit peptidecomprises a mitochondrial transit peptide.

E6. The composition of embodiment 5, wherein the mitochondrial transitpeptide is derived from a protein selected from the group consisting ofSEQ ID NOs: 251 to 265 and 270 to 719.

E7. The composition of any one of embodiments 4 to 6, wherein thenucleic acid sequence encoding a transit peptide has at least about 70%,at least about 80%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%,or about 100% sequence identity to a sequence selected from the groupconsisting of nucleotides 1 to 96 of SEQ ID NOs: 1 to 207, 732 to 765,and 772.

E8. The composition of any one of embodiments 1 to 7, wherein the ORFhas

(i) at least 99% or 100% sequence identity to nucleotide 1 to nucleotide2250 of SEQ ID NO: 734;

(ii) at least 98%, at least 99%, or 100% sequence identity to nucleotide1 to nucleotide 2250 of SEQ ID NO: 732;

(iii) at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identityto a sequence selected from the group consisting of nucleotides 1 tonucleotides 2250 of SEQ ID NOs: 182 and 733;

(iv) at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a sequence selected from the group consisting ofnucleotides 1 to nucleotides 2250 of SEQ ID NOs: 735, 741, 743, 744,748, 758, 762, and 765;

(v) at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% sequence identity to a sequence selected from the groupconsisting of nucleotides 1 to nucleotides 2250 of SEQ ID NOs: 180, 181,736, 738, 739, 740, 742, 746, 747, 749, 750, 751, 752, 753, 754, 755,757, 759, 760, 761, and 763;

(vi) at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to a sequence selected fromthe group consisting of nucleotides 1 to nucleotides 2250 of SEQ ID NO:745, 756, and 764;

(vii) at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% sequence identity to a sequenceselected from the group consisting of nucleotides 1 to nucleotides 2250of SEQ ID NO: 154, 165, 171, 173, and 175;

(viii) at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identityto a sequence selected from the group consisting of nucleotides 1 tonucleotides 2250 of SEQ ID NO: 151, 152, 153, 163, 166, 167, 168, 169,170, 172, 177, 178, 179, 187, and 204;

(ix) at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a sequence selected from the group consisting ofnucleotides 1 to nucleotides 2250 of SEQ ID NO: 156, 157, 158, 159, 160,162, 164, 174, 176, 195, and 737;

(x) at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% sequence identity to a sequence selected from the groupconsisting of nucleotides 1 to nucleotides 2250 of SEQ ID NOs: 155, 161,and 203;

(xi) at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identityto a sequence selected from the group consisting of nucleotides 1 tonucleotides 2250 of SEQ ID NOs: 71 and 128;

(xii) at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a sequence selected from the group consisting ofnucleotides 1 to nucleotides 2250 of SEQ ID NOs: 4, 6, 8, 9, 11, 19, 22,23, 24, 32, 33, 37, 40, 44, 45, 47, 51, 61, 64, 65, 66, 79, 84, 86, 90,91, 92, 100, 101, 112, 115, 117, 126, 129, 135, 136, 146, 148, 184, 190,and 191;

(xiii) at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% sequence identity to a sequence selected from the groupconsisting of nucleotides 1 to nucleotides 2250 of SEQ ID NOs: 2, 3, 5,7, 10, 12, 13, 14, 15, 16, 18, 20, 21, 26, 27, 28, 29, 31, 34, 36, 38,39, 41, 42, 43, 46, 48, 49, 52, 53, 54, 55, 56, 57, 58, 59, 60, 62, 68,69, 70, 72, 73, 74, 76, 77, 80, 83, 85, 88, 95, 96, 97, 98, 102, 104,105, 106, 107, 108, 109, 110, 113, 114, 120, 121, 122, 123, 124, 127,131, 132, 133, 134, 137, 138, 139, 140, 141, 142, 144, 145, 147, 149,150, 186, 188, 189, 192, 193, 194, 196, 198, 199, 200, 202, 205, 206,and 207; or

(xiv) at least 79%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to a sequence selected fromthe group consisting of nucleotides 1 to nucleotides 2250 of SEQ ID NOs:1, 17, 25, 30, 35, 50, 63, 67, 75, 78, 81, 82, 87, 89, 93, 94, 99, 103,111, 116, 118, 119, 125, 130, 143, 183, 185, 197, and 201.

E9. The composition of any one of embodiments 1 to 8, wherein the MCMpolypeptide comprises an amino acid sequence at least about 95%, atleast about 96%, at least about 97%, at least about 98%, at least about99%, or about 100% identical to SEQ ID NO: 208, and wherein the MCMpolypeptide retains methylmalonyl-CoA mutase activity.

E10. The composition of embodiment 9, wherein the MCM polypeptidecomprises SEQ ID NO: 209.

E11. The composition of embodiment 9, wherein the MCM polypeptidecomprises SEQ ID NO: 210.

E12. The composition of embodiment 9, wherein the MCM polypeptidecomprises SEQ ID NO: 211.

E13. The composition of embodiment 9, wherein the MCM polypeptidecomprises SEQ ID NO: 212.

E14. The composition of embodiment 9, wherein the MCM polypeptidecomprises SEQ ID NO: 213.

E15. The composition of any one of embodiments 1-14, wherein thepolynucleotide comprises at least one chemically modified nucleobase,sugar, backbone, or any combination thereof.

E16. The composition of embodiment 15, wherein the at least onechemically modified nucleobase is selected from the group consisting ofpseudouracil (ψ), N1-methylpseudouracil (m1ψ), 2-thiouracil (s2U),4′-thiouracil, 5-methylcytosine, 5-methyluracil, and any combinationthereof.

E17. The composition of embodiment 16, wherein the at least onechemically modified nucleoside is 5-methoxyuracil.

E18. The composition of any one of embodiments 1-17, wherein thenucleosides in the polynucleotide sequence are chemically modified by atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 99%, or 100%.

E19. The composition of any one of embodiments 15-18, wherein thechemically modified nucleosides in the polynucleotide sequence areselected from the group consisting of uridine, adenine, cytosine,guanine, and any combination thereof.

E20. The composition of any one of embodiments 1-19, wherein the uridinenucleosides in the polynucleotide sequence are chemically modified by atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 99%, or 100%.

E21. The composition of any one of embodiments 1-20, wherein the adeninenucleosides in the polynucleotide sequence are chemically modified by atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 99%, or 100%.

E22. The composition of any one of embodiments 1-21, wherein thecytosine nucleosides in the polynucleotide sequence are chemicallymodified by at least at least 10%, at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 99%, or 100%.

E23. The composition of any one of embodiments 1-22, wherein the guaninenucleosides in the polynucleotide sequence are chemically modified by atleast at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 99%, or 100%.

E24. The composition of any one of embodiments 1-23, wherein thepolynucleotide further comprises a 5′ UTR.

E25. The composition of embodiment 24, wherein the 5′ UTR comprises anucleic acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to a sequence selected from SEQ ID NOs: 215-231, 266, and725-731.

E26. The composition of any one of embodiments 1 to 25, wherein thepolynucleotide further comprises a miRNA binding site.

E27. The composition of embodiment 26, wherein the miRNA binding sitecomprises one or more polynucleotide sequences selected SEQ ID NOs: 720,721, 722, 723, and 724.

E28. The composition of embodiment 26, wherein the miRNA binding sitebinds to miR-142 or miR-126.

E29. The composition of embodiment 26, wherein the miRNA binding sitebinds to miR-142-3p, miR-142-5p, miR-126-3p, or miR-126-5p.

E30. The composition of embodiment 24, wherein the 5′UTR comprises asequence selected from SEQ ID NOs: 725, 726, 727, 728, 729, 730, and731.

E31. The composition of any one of embodiments 24-30, wherein the 5′ UTRis sequence optimized.

E32. The composition of any one of embodiments 1-31, wherein thepolynucleotide further comprises a 3′ UTR.

E33. The composition of embodiment 32, wherein the 3′ UTR comprises anucleic acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to a sequence selected from SEQ ID NO: 232-248 and 267.

E34. The composition of embodiment 32 or 33, wherein the 3′ UTR is codonoptimized.

E35. The composition of any one of embodiments 1-34, wherein thepolynucleotide further comprises a 5′ terminal cap.

E36. The composition of embodiment 35, wherein the 5′ terminal cap is aCap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2′fluoro-guanosine,7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine,2-azidoguanosine, Cap2, Cap4, 5′ methylG cap, or an analog thereof.

E37. The composition of any one of embodiments 1-36, wherein thepolynucleotide further comprises a 3′ polyA tail.

E38. The composition of any one of embodiments 1-37, wherein thepolynucleotide is RNA.

E39. The composition of embodiment 38, wherein the RNA is mRNA.

E40. The composition of any one of embodiments 1-39, wherein thepolynucleotide is in vitro transcribed (IVT).

E41. The composition of any one of embodiments 1-40, wherein thepolynucleotide is chimeric.

E42. The composition of any one of embodiments 1-41, wherein thepolynucleotide is circular.

E43. The composition of any one of embodiments 1-42, wherein thepolynucleotide is purified by strong anion exchange HPLC, weak anionexchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interactionHPLC (HIC-HPLC), liquid chromatography-mass spectrometry (LCMS),capillary electrophoresis (CE) and capillary gel electrophoresis (CGE).

E44. The composition of any one of embodiments 1-43, wherein thecompound is of Formula (IA):

or a salt or stereoisomer thereof, wherein

l is selected from 1, 2, 3, 4, and 5;

m is selected from 5, 6, 7, 8, and 9;

M₁ is a bond or M′;

R₄ is unsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which n is 1, 2, 3,4, or 5 and Q is OH, —NHC(S)N(R)₂, or —NHC(O)N(R)₂;

M and M′ are independently selected from C(O)O, OC(O), C(O)N(R′),P(O)(OR′)O, an aryl group, and a heteroaryl group; and

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

E45. The composition of any one of embodiments 1 to 44, wherein m is 5,7, or 9.

E46. The composition of any one of embodiments 1 to 45, wherein thecompound is of Formula (II):

or a salt or stereoisomer thereof, wherein

l is selected from 1, 2, 3, 4, and 5;

M₁ is a bond or M′;

R₄ is unsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which n is 2, 3, or 4and Q is OH, —NHC(S)N(R)₂, or —NHC(O)N(R)₂;

M and M′ are independently selected from —C(O)O, OC(O), C(O)N(R′),P(O)(OR′)O, an aryl group, and a heteroaryl group; and

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

E47. The composition of any one of embodiments 44 to 46, wherein M₁ isM′.

E48. The composition of embodiment 47, wherein M and M′ areindependently —C(O)O— or —OC(O)—.

E49. The composition of any one of embodiments 44 to 48, wherein 1 is 1,3, or 5.

E50. The composition of any one of embodiments 1 to 43, wherein thecompound is selected from the group consisting of Compound 1 to Compound147, salts and stereoisomers thereof, and any combination thereof.

E51. The composition of any one of embodiments 1 to 43, wherein thecompound is of the Formula (IIa),

or a salt or stereoisomer thereof.

E52. The composition of any one of embodiments 1 to 43, wherein thecompound is of the Formula (IIb),

or a salt or stereoisomer thereof.

E53. The composition of any one of embodiments 1 to 43, wherein thecompound is of the Formula (IIc) or (IIe),

or a salt or stereoisomer thereof.

E54. The composition of any one of embodiments 51 to 53, wherein R₄ isselected from —(CH₂)_(n)Q and —(CH₂)_(n)CHQR.

E55. The composition of any one of embodiments 1 to 43, wherein thecompound is of the Formula (IId),

or a salt or stereoisomer thereof,

wherein R₂ and R₃ are independently selected from the group consistingof C₅₋₁₄ alkyl and C₅₋₁₄ alkenyl, n is selected from 2, 3, and 4, andR′, R″, R₅, R₆ and m are as defined in embodiment 1.

E56. The composition of embodiment 55, wherein R₂ is C₈ alkyl.

E57. The composition of embodiment 56, wherein R₃ is C₅ alkyl, C₆ alkyl,C₇ alkyl, C₈ alkyl, or C₉ alkyl.

E58. The composition of any one of embodiments 55 to 57, wherein m is 5,7, or 9.

E59. The composition of any one of embodiments 55 to 58, wherein each R₅is H.

E60. The composition of embodiment 59, wherein each R₆ is H.

E61. The composition of any one of embodiments 1 to 60, wherein thecomposition is a nanoparticle composition.

E62. The composition of embodiment 61, wherein the delivery agentfurther comprises a phospholipid.

E63. The composition of embodiment 62, wherein the phospholipid isselected from the group consisting of1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC),1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC),1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine(OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),1,2-dilinolenoyl-sn-glycero-3-phosphocholine,1,2-diarachidonoyl-sn-glycero-3-phosphocholine,1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine,1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16:0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine,1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine,1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine,1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG),sphingomyelin, and any mixtures thereof.

E64. The composition of any one of embodiments 1 to 63, wherein thedelivery agent further comprises a structural lipid.

E65. The composition of embodiment 64, wherein the structural lipid isselected from the group consisting of cholesterol, fecosterol,sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol,tomatidine, ursolic acid, alpha-tocopherol, and any mixtures thereof.

E66. The composition of any one of embodiments 1 to 65, wherein thedelivery agent further comprises a PEG lipid.

E67. The composition of embodiment 66, wherein the PEG lipid is selectedfrom the group consisting of a PEG-modified phosphatidylethanolamine, aPEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modifieddialkylamine, a PEG-modified diacylglycerol, a PEG-modifieddialkylglycerol, and any mixtures thereof.

E68. The composition of any one of embodiments 1 to 67, wherein thedelivery agent further comprises an ionizable lipid selected from thegroup consisting of3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10),N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine(KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25),1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate(DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA),(2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2R)), and(2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2S)).

E69. The composition of any one of embodiments 1 to 68, wherein thedelivery agent further comprises a phospholipid, a structural lipid, aPEG lipid, or any combination thereof.

E70. The composition of any one of embodiments 1-69, wherein thecomposition is formulated for in vivo delivery.

E71. The composition of embodiment 70 which is formulated forintramuscular, subcutaneous, or intradermal delivery.

E72. The composition of any one of embodiments 1-71 which increasescellular expression of MCM.

E73. The composition of embodiment 72, wherein the cellular expressionof MCM is increased by at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, or at least 50%.

E74. A method of administering the composition of any one of embodiments1-73, wherein the administration alleviates the symptoms ofmethylmalonic acidemia in the subject.

E75. The method of embodiment 74, wherein the administration results ina reduction of the level of a metabolite associated with methylmalonicacidemia in a subject in need thereof.

E76. The method of embodiment 74 or 75, further comprising measuring thelevel of the metabolite in the subject or in a sample obtained from thesubject before and/or after the administering.

E77. The method of embodiment 76, wherein the sample is taken from thesubject's blood, urine, cerebrospinal fluid, or any combination thereof.

E78. The method of any of embodiments 74 to 77, wherein theadministration reduces the level of methylmalonic acid present in thesubject by at least about 10%, at least about 20%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, or about 100%.

E79. The method of any of embodiments 74 to 78, wherein thepolynucleotide reduces the level of propionyl-carnitine present in thesubject by at least about 10%, at least about 20%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, or about 100%.

E80. The method of any of embodiments 74 to 79, wherein thepolynucleotide reduces the level of acetyl-carnitine present in thesubject by at least about 10%, at least about 20%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, or about 100%.

E81. The method of any one of embodiments 74 to 80, wherein one or moremetabolites associated with methylmalonic acidemia are reduced withinone day, within two days, within three days, within four days, withinfive days, within seven days, within one week, within two weeks, withinthree weeks, or within one month of the administration of thepolynucleotide.

E82. A method of treating methylmalonic acidemia in a subject in needthereof, the method comprising administering to the subject an effectiveamount of a polynucleotide comprising an mRNA that comprises an ORFencoding an MCM polypeptide, wherein the administration alleviates thesymptoms of methylmalonic acidemia in the subject.

E83. The method of embodiment 82, wherein the polynucleotide comprisesthe polynucleotide in the composition of any one of embodiments 1 to 73.

E84. The method of embodiment 82 or 83, wherein the administrationreduces the level of a metabolite associated with methylmalonic acidemiain a subject in need thereof.

E85. The method of any one of embodiments 82-84, further comprisingmeasuring the level of the metabolite in the subject or in a sampleobtained from the subject before and/or after the administering.

E86. The method of embodiment 85, wherein the sample is taken from thesubject's blood, urine, cerebrospinal fluid, or any combination thereof.

E87. The method of any of embodiments 82 to 86, wherein thepolynucleotide reduces the level of methylmalonic acid present in thesubject by at least about 10%, at least about 20%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, or about 100%.

E88. The method of any of embodiments 82 to 87, wherein thepolynucleotide reduces the level of propionyl-carnitine present in thesubject by at least about 10%, at least about 20%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, or about 100%.

E89. The method of any of embodiments 82 to 88, wherein thepolynucleotide reduces the level of acetyl-carnitine present in thesubject by at least about 10%, at least about 20%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, or about 100%.

E90. The method of any one of embodiments 82 to 89, wherein one or moremetabolites associated with methylmalonic acidemia are reduced withinone day, within two days, within three days, within four days, withinfive days, within seven days, within one week, within two weeks, withinthree weeks, or within one month of the administration of thepolynucleotide.

E91. The method of any one of embodiments 82 to 90, wherein thenucleotide is administered as a nanoparticle composition.

E92. The method of embodiment 91, wherein the composition furthercomprises a delivery agent.

E93. The method of embodiment 92, wherein the delivery agent comprises aphospholipid.

E94. The method of embodiment 93, wherein the phospholipid is selectedfrom the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine(DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC),1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine(OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),1,2-dilinolenoyl-sn-glycero-3-phosphocholine,1,2-diarachidonoyl-sn-glycero-3-phosphocholine,1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine,1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16:0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine,1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine,1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine,1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG),sphingomyelin, and any mixtures thereof.

E95. The method of any one of embodiments 92 to 94, wherein the deliveryagent further comprises a structural lipid.

E96. The method of embodiment 95, wherein the structural lipid isselected from the group consisting of cholesterol, fecosterol,sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol,tomatidine, ursolic acid, alpha-tocopherol, and any mixtures thereof.

E97. The method of any one of embodiments 92 to 96, wherein the deliveryagent further comprises a PEG lipid.

E98. The method of embodiment 97, wherein the PEG lipid is selected fromthe group consisting of a PEG-modified phosphatidylethanolamine, aPEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modifieddialkylamine, a PEG-modified diacylglycerol, a PEG-modifieddialkylglycerol, and any mixtures thereof.

E99. The method of any one of embodiments 92 to 98, wherein the deliveryagent further comprises an ionizable lipid selected from the groupconsisting of3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10),N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine(KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25),1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate(DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA),(2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2R)), and(2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2S)).

E100. The method of any one of embodiments 92 to 99, wherein thedelivery agent further comprises a phospholipid, a structural lipid, aPEG lipid, or any combination thereof.

E101. The method of any one of embodiments 92-100, wherein thecomposition is formulated for in vivo delivery.

E102. The method of embodiment 101 which is formulated forintramuscular, subcutaneous, or intradermal delivery.

E103. The method of any one of embodiments 82-102 which increasescellular expression of MCM.

E104. The method of embodiment 103, wherein the cellular expression ofMCM is increased by at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, or at least 50%.

E105. The method of any one of embodiments 74 to 104, wherein thepolynucleotide is administered at a dose of 0.1 mg/kg to 1.0 mg/kg, 0.1mg/kg to 10 mg/kg, 0.1 mg/kg to 2 mg/kg, 0.1 mg/kg to 5 mg/kg, 1 mg/kgto 5 mg/kg, or 1 mg/kg to 3 mg/kg.

E106. The method of any one of embodiments 74 to 105, wherein the plasmaMMA level after the administration is reduced at least 70%, at least75%, at least 80%, at least 85%, at least 90%, or at least 95% comparedto the plasma MMA level prior to the administration.

E107. The method of embodiment 106, wherein the plasma MMA level isreduced about 75% to 85% compared to the plasma MMA level prior to theadministration.

E108. The method of any one of embodiments 74 to 107, wherein the plasmaMMA level after the administration is lower than about 5 μmol/L, about4.5 μmol/L, about 4 μmol/L, about 3.5 μmol/L, about 3 μmol/L, about 2.5μmol/L, about 2 μmol/L, about 1.5 μmol/L, about 1 μmol/L, about 0.9μmol/L, about 0.8 μmol/L, about 0.7 μmol/L, about 0.6 μmol/L, about 0.5μmol/L, about 0.4 μmol/L, about 0.3 μmol/L, or 0.27 μmol/L.

E109. The method of embodiments 74 to 108, wherein the urinary MMA levelis less than 2000 mmol/mol creatinine, less than 1900 mmol/molcreatinine, less than 1800 mmol/mol creatinine, less than 1700 mmol/molcreatinine, less than 1600 mmol/mol creatinine, less than 1500 mmol/molcreatinine, less than 1400 mmol/mol creatinine, less than 1300 mmol/molcreatinine, less than 1200 mmol/mol creatinine, less than 1100 mmol/molcreatinine, less than 1000 mmol/mol creatinine, 900 mmol/mol creatinine,800 mmol/mol creatinine, 700 mmol/mol creatinine, 600 mmol/molcreatinine, 500 mmol/mol creatinine, 400 mmol/mol creatinine, 300mmol/mol creatinine, 200 mmol/mol creatinine, 100 mmol/mol creatinine,90 mmol/mol creatinine, 80 mmol/mol creatinine, 70 mmol/mol creatinine,60 mmol/mol creatinine, 50 mmol/mol creatinine, 40 mmol/mol creatinine,30 mmol/mol creatinine, 20 mmol/mol creatinine, 10 mmol/mol creatinine,9 mmol/mol creatinine, 8 mmol/mol creatinine, 7 mmol/mol creatinine, 6mmol/mol creatinine, 5 mmol/mol creatinine, 4 mmol/mol creatinine, 3mmol/mol creatinine, 2 mmol/mol creatinine, or 1 mmol/mol creatinine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Western blot analysis of endogenous methylmalonyl-CoA mutaseexpression in a mouse liver mitochondrial extract, mouse cells(Hepa1-6), and human cells (HepG2, SNU423, and HeLa). The upper band(thin arrow) shows Mouse α-Methymalonyl-CoA mutase, and the lower band(thick arrow) shows Rabbit α-Citrate synthetase.

FIGS. 2A-2C show immunofluorescence analyses of the localization ofendogenous methylmalonyl-CoA mutase in HeLa cells. FIG. 2A shows thelocation of mitochondria using Mitotracker and the nucleus using DAPI.FIG. 2B shows the immunostaining of hMCM protein using a murinemonoclonal anti-MCM antibody and the location of the nucleus using DAPI.FIG. 2C shows the merged picture of FIGS. 2A and 2B.

FIG. 3 is a Western blot analysis comparing methylmalonyl-CoA mutaseexpression in (i) HeLa cells transfected with a control GFP expressionconstruct, (ii) HeLa cells transfected with a construct for expressingmethylmalonyl-CoA mutase, and (iii) a mouse liver mitochondrial extract.

FIG. 4 is a comparison of methylmalonyl-CoA mutase enzymatic activity in(i) HeLa cells transfected with a control GFP expression construct, (ii)HeLa cells transfected with a construct for expressing methylmalonyl-CoAmutase, and (iii) a mouse liver mitochondrial extract.

FIG. 5 is a Western blot analysis of methylmalonyl-CoA mutase expressionin Hepa1-6 cells, fibroblasts from normal human subjects (NHDF), andfibroblasts from MMA patients (GM50 and GM1573) that were transfectedwith control mRNA, human MCM mRNA, or mouse MCM mRNA.

FIGS. 6A-6D show immunofluorescence analyses of the localization ofexogenously expressed methylmalonyl-CoA mutase in human fibroblaststransfected with eGFP mRNAs or MCM mRNAs (also referred to as “MUT”).The left panels show the location of mitochondria using Mitotracker andthe nucleus using DAPI, the middle panels show shows the location ofmitochondria using MCM protein and the nucleus using DAPI, and the rightpanels show merged images of the left panel and the right panel. FIGS.6A and 6C are images taken of patient fibroblasts transfected with mRNAencoding eGFP. FIGS. 6B and 6D are images taken of patient fibroblaststransfected with mRNA encoding hMCM.

FIG. 7 is measurement of methylmalonyl-CoA mutase activity in Hepa1-6cells, fibroblasts from normal human subjects (NHDF), and fibroblastsfrom MMA patients (GM50 and GM1573) that were transfected with controlmRNA, human MCM mRNA, or mouse MCM mRNA.

FIGS. 8A-8B is an analysis of in vivo treatment with mRNA encodingmethylmalonyl-CoA mutase. For FIGS. 8A and 8B, C57B/L6 mice wereinjected intravenously with either control mRNA (NT-FIX) or MCM mRNA at0.5 mg mRNA/kg body weight (“mpk”). Mice were sacrificed after 24 or 48hours and MCM protein in mitochondria from livers were determined bycapillary electrophoresis (CE). The upper panel (FIG. 8A) showsinjection of MCM mRNA increased MCM protein expression after 24 and 48hours, while the lower panel (FIG. 8B) shows the expression of thecontrol protein citrate synthase.

FIGS. 9-15 show exemplary codon optimized MCM sequences that encodemethylmalonyl-CoA mutase. The illustrated sequences in FIGS. 9-15 areSEQ ID NOs: 732, 733, 734, 151, 152, 153, and 154, respectively.

FIGS. 16A-C show analysis of MMA levels and body weight in the MCK mousemodel. FIG. 16A shows plasma levels of methylmalonic acid (MMA) in μMmeasured by LC-MS/MS over time in mice treated weekly with control mRNA(NT-FIX) at 0.1 mg/kg, codon optimized MCM mRNA (encoding SEQ ID NO:734)formulated in lipid nanoparticles at 0.16 or 0.2 mg/kg for 5 injections,or codon optimized MCM mRNA (encoding SEQ ID NO:734) formulated in lipidnanoparticles at 0.2 mg/kg for 2 injections. FIG. 16B shows the bodyweight of the mice over time (measured twice a week). ***p<0.001;P-values obtained from repeated measures ANOVA. FIG. 16C shows theincrease in body weight over time in mice injected weekly with codonoptimized MCM mRNA.

FIG. 17 shows MCM expression in liver of wild-type CD1 mice dosed withcodon optimized MCM mRNA (SEQ ID NO: 734) formulated in lipidnanoparticles at 0.2 mg/kg compared to endogenous human MCM andendogenous mouse MCM.

FIGS. 18A-C show a time course of the effects of injection of codonoptimized MCM mRNA. FIG. 18A shows levels of lipid nanoparticles aftersingle dose injection of codon optimized MCM mRNA. FIG. 18B showsHepatic hMut mRNA levels in mouse liver after single dose injection ofcodon optimized MCM mRNA. FIG. 18C shows MCM protein levels after singledose injection of codon optimized MCM mRNA

FIGS. 19A-B show MCM expression in livel of wild-type CD mice dosed withcodon optimized MCM mRNAs, where the mRNA is formulated either with MC3or Compound 18. NTFIX mRNA was used as a control. FIG. 19A shows aWestern blot of expression after dosing with different formulations, andFIG. 19B shows a quantification of that Western blot.

FIGS. 20A-B show the effects of administering codon optimized MCM mRNAto mice on the plasma levels of MMA (FIG. 20A) and the body weight ofthe mice (FIG. 20B).

DETAILED DESCRIPTION

The present disclosure provides polynucleotide sequences that encode asequence-optimized nucleic acid encoding a methylmalonyl-CoA mutasepolypeptide (“MCM” or “MUT”). MCM is the principal gene associated withmethylmalonic acidemia (“MMA,” also referred to as methylmalonicadicuria). Wild type nucleic acid and amino acid sequences for humanmethylmalonyl-CoA mutase (MCM) are described in NCBI sequence recordsgi296010795 (reference sequence NM_000255.3, “Homo sapiensmethylmalonyl-CoA mutase (MUT), mRNA”; see also, SEQ ID NO: 214) andgi156105689 (reference sequence NP_000246.2, “methylmalonyl-CoA mutase,mitochondrial precursor [Homo sapiens]”; see also, SEQ ID NO: 208),respectively. Accession numbers and the associated sequences are foundat the National Center for Biotechnology Information (NCBI) website.

MCM is a metabolic enzyme (E.C. 5.4.99.2), the biological function ofwhich is to isomerize L-methylmalonyl-CoA into succinyl-CoA, a Krebscycle intermediate. MCM localizes to the mitochondria of cells, existsas a homodimer in its native form, and is adenosylcobalamin-dependent.The precursor form of human MCM is 750 amino acids, while its matureform is 718 amino acids—a 32 amino acid leader sequence is cleaved offby mitochondrial importation and processing machinery.

I. COMPOSITION Polynucleotides Encoding MCM

In certain aspects, the present disclosure provides nucleic acidmolecules, specifically polynucleotides that encode one or more MCMpolypeptides. The MCM polypeptides that are encoded can be mammalian MCMpolypeptides, for example, human MCM peptides, or functional fragmentsthereof.

In some embodiments, the polynucleotides described herein encode atleast one methylmalonyl-CoA mutase protein, functional fragment, orvariant thereof. MCM catalyzes enzymatic transformation ofmethylmalonyl-CoA into succinyl-CoA, and also comprises acobalamin-binding domain. MCM's enzymatic activity is dependent on itsbinding to its cofactor, denosylcobalamin.

MCM plays a critical role in the catabolism of fat and protein,specifically in disposing of methylmalonyl-CoA created duringmetabolism. For example, methylmalonyl-CoA is an intermediate in thecatabolism of amino acids such as isoleucine, methionine, and threonine.Methylmalonyl-CoA is also an intermediate in the catabolism ofcholesterol and fatty acids. Defects in the activity of this enzyme leadto inefficient metabolism and buildup of potentially toxic metabolicintermediates such as methylmalonic acid. The lack of MCM causes thedisorder known as methylmalonic acidemia (MMA).

Replacement of MCM has been theorized to be a cure of this form of MMA.In some embodiments, the polynucleotides disclosed herein comprise oneor more sequences encoding a methylmalonyl-CoA mutase protein,functional fragment, or variant thereof that is suitable for use in suchgene replacement therapy. In certain aspects, the present applicationaddresses the problem of the lack of methylmalonyl-CoA mutase byproviding a polynucleotide, e.g., mRNA, that encodes methylmalonyl-CoAmutase or functional fragment thereof, wherein the polynucleotide issequence-optimized. In some embodiments, the polynucleotide, e.g., mRNA,increases MCM expression levels in cells when introduced into thosecells, e.g., by at least 20%, at least 20%, at least 25%, at least 35%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, or at least 100%.

In some embodiments, the polynucleotides of the disclosure encodefunctional MCM polypeptides or fragments thereof. In some embodiments,the polynucleotides of the disclosure encode an MCM protein or variantthereof that is full length (i.e., it includes a mitochondrial transitpeptide, either native or heterologous to that in native full-lengthMCM), while in other embodiments polynucleotides of the disclosureencode a functional MCM protein or variant thereof that is mature (i.e.,it lacks the mitochondrial transit peptide). In some embodiments, thepolynucleotides encode a human MCM, or variant thereof, linked to aheterologous or homologous mitochondrial transit peptide.

In some embodiments, the polynucleotides of the disclosure encodefunctional human MCM (SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210,SEQ ID NO: 211, SEQ ID NO: 212, and SEQ ID NO: 213) or fragmentsthereof. In some embodiments, the polynucleotides of the disclosureencode mutant MCM. In some embodiments, the polynucleotides encode anMCM polypeptide that comprises at least one point mutation in the MCMsequence, while still retaining MCM enzymatic activity. In someembodiments, the polynucleotides encode a functional MCM polypeptidewith mutations that do not alter the function of MCM. Such functionalMCM can be referred to as function-neutral. In some embodiments, theencoded MCM polypeptide comprises one or more of the function-neutralpoint mutations V69, T499, H532, A598, and V671. In some embodiments,the polynucleotides of the disclosure encode the polypeptide sequencesin SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, andSEQ ID NO: 213, which contain the function-neutral mutants V69, T499,H532, A598, and V671, respectively. In particular embodiments, theencoded MCM polypeptide is a V671 mutant (SEQ ID NO: 213).Polynucleotides encoding MCM polypeptides are listed in SEQ ID NOs: 1 to207, 214, 732 to 765, and 772.

In some embodiments, the polynucleotides comprise a nucleotide sequencehaving significant sequence similarity to a polynucleotide selected fromthe group of SEQ ID NOs: 1-207, 732-765, and 772, wherein the ORFencodes an MCM polypeptide. In some embodiments, the polynucleotidecomprises a nucleotide sequence having significant sequence similarityto SEQ ID NOs: 151, 152, 153, 154, 732, 733, and 734 (FIGS. 9-15). Insome embodiments, the polynucleotide comprises a nucleotide sequencehaving significant sequence similarity to SEQ ID NO: 734 (FIG. 11).

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 99% or 100% sequence identity tonucleotide 97 to nucleotide 2250 of SEQ ID NO: 734, wherein the ORFencodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 98%, at least 99%, or 100% sequenceidentity to nucleotide 97 to nucleotide 2250 of SEQ ID NO: 732, whereinthe ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to a sequence selected from the group consistingof nucleotides 97 to nucleotides 2250 of SEQ ID NOs: 182, 733, and 741,wherein the ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to a sequence selected from thegroup consisting of nucleotides 97 to nucleotides 2250 of SEQ ID NOs:735, 736, 738, 743, 744, 748, 749, 750, 754, 755, 758, 762, and 765,wherein the ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to a sequenceselected from the group consisting of nucleotides 97 to nucleotides 2250of SEQ ID NOs: 180, 187, 737, 739, 740, 742, 745, 746, 747, 751, 752,753, 757, 759, 760, 761, 763, and 764, wherein the ORF encodes an MCMpolypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to asequence selected from the group consisting of nucleotides 97 tonucleotides 2250 of SEQ ID NO: 181 and 756, wherein the ORF encodes anMCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to a sequence selected from the group consisting of nucleotides97 to nucleotides 2250 of SEQ ID NO: 154, 165, 171, 173, and 175,wherein the ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to a sequence selected from the group consistingof nucleotides 97 to nucleotides 2250 of SEQ ID NO: 151, 152, 153, 163,164, 166, 167, 168, 169, 170, 172, 177, 178, 179, 195, and 204, whereinthe ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to a sequence selected from thegroup consisting of nucleotides 97 to nucleotides 2250 of SEQ ID NOs:156, 157, 158, 159, 160, 161, 162, 174 and 176, wherein the ORF encodesan MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to a sequenceselected from the group consisting of nucleotides 97 to nucleotides 2250of SEQ ID NOs: 155 and 203, wherein the ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to a sequence selected from the group consistingof nucleotides 97 to nucleotides 2250 of SEQ ID NOs: 64, 66, 71, 91, and128, wherein the ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to a sequence selected from thegroup consisting of nucleotides 97 to nucleotides 2250 of SEQ ID NOs: 9,11, 18, 19, 21, 22, 23, 24, 32, 33, 37, 39, 40, 44, 45, 47, 50, 51, 52,55, 57, 61, 65, 70, 79, 84, 86, 88, 90, 92, 98, 100, 115, 117, 126, 129,135, 136, 137, 144, 148, 150, 184, 190, 191, and 206, wherein the ORFencodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to a sequenceselected from the group consisting of nucleotides 97 to nucleotides 2250of SEQ ID NOs: 3, 5, 6, 8, 10, 12, 14, 16, 17, 20, 27, 28, 29, 31, 34,35, 36, 38, 41, 42, 43, 46, 48, 49, 53, 54, 56, 58, 60, 63, 68, 69, 74,77, 78, 80, 83, 85, 87, 93, 95, 96, 97, 99, 102, 103, 104, 105, 107,110, 112, 113, 114, 116, 119, 120, 122, 123, 124, 125, 127, 131, 132,133, 134, 138, 139, 140, 141, 142, 143, 147, 149, 183, 186, 188, 189,192, 193, 194, 196, 197, 198, 199, 200, 201, 202, 205, and 207, whereinthe ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 79%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to asequence selected from the group consisting of nucleotides 97 tonucleotides 2250 of SEQ ID NOs: 1, 2, 4, 7, 13, 15, 25, 26, 30, 59, 62,67, 72, 73, 75, 76, 81, 82, 89, 94, 101, 106, 108, 109, 111, 118, 121,130, 145, 146, and 185, wherein the ORF encodes an MCM polypeptide.

The polynucleotides of the disclosure can also encode additionalfeatures that facilitate trafficking of the polypeptides totherapeutically relevant sites. One such feature that aids in proteintrafficking is the signal sequence, or targeting sequence. The peptidesencoded by these signal sequences are known by a variety of names,including targeting peptides, transit peptides, and signal peptides. Thedisclosure also includes a polynucleotide comprising a sequence thatencodes a mitochondrial transit peptide operably linked to thepolynucleotide described herein, i.e., linked to a polynucleotidecomprising an ORF encoding an MCM polypeptide.

As used herein, a “signal sequence” or “signal peptide” is apolynucleotide or polypeptide, respectively, which is from about 9 to200 nucleotides (3-70 amino acids) in length that, in some embodiments,is incorporated at the 5′ (or N-terminus) of the coding region orpolypeptide encoded, respectively. Addition of these sequences result intrafficking of the encoded polypeptide to a desired site, such as theendoplasmic reticulum or the mitochondria through one or more targetingpathways. Some signal peptides are cleaved from the protein, for exampleby a signal peptidase after the proteins are transported.

For example, human MCM's precursor protein comprises a 32-amino acidmitochondrial transit peptide, also referred to as an MCM mitochondrialtargeting sequence or mitochondrial targeting peptide, that facilitatesdelivery of the MCM protein to, and localization in, mitochondria. Thepresent disclosure comprises both polynucleotides that encode ahomologous targeting sequence (i.e., MCM's native mitochondrial transitsequence) and polynucleotides that encode a heterologous mitochondrialtransit sequences (i.e., a mitochondrial transit peptide that is not thenative targeting peptide for the operably linked MCM protein). In someembodiments, the alternate targeting sequences facilitate delivery ofMCM to mitochondria.

Exemplary sequences of known mitochondrial transit peptides includeMLSLRQSIRFFKPATRTLCSSRYLL (SEQ ID NO: 251), MALLRGVFVVAAKRTP (SEQ ID NO:252) and MLRIPVRKALVGLSKSSKGCVRT (SEQ ID NO: 253). Non-limiting examplesof the mitochondrial transit peptides are listed below in Table 1 (SEQID NOs: 251-265). Further examples of mitochondrial transit peptides areprovided as SEQ ID NOs: 270-719. Additional mitochondrial transitpeptides that can be utilized in the present disclosure can beidentified using predictive tools known in the art. For example,mitochondrial targeting can be analyzed using the methods described inFukusawa et al., Molecular and Cellular Proteomics 14:1113-1126 (2015),the contents of which are incorporated herein in their entirety.

TABLE 1 Mitochondrial Transit Peptides ID (SEQ ID NO)Name of the protein Sequence COX4 Saccharomyces cerevisiae mitochondrialMLSLRQSIRFFKPATRTLCSSRYLL (SEQ ID NO: cytochrome c oxidase subunit IV251) ACAA2 Mitochondrial 3-ketoacyl-coa thiolase MALLRGVFVVAAKRTP(SEQ ID NO: 252) NDUFS1 NADH-ubiquinone oxidoreductase 75 kDaMLRIPVRKALVGLSKSSKGCVRT (SEQ ID NO: subunit, mitochondrial isoform 2253) A6NK58 Putative lipoyltransferase 2, mitochondrial (ECMRQPAVRLVRLGRVPYAELLGLQDRWLR (SEQ ID NO:2.3.1.181) (Lipoate-protein ligase B) RLQ 254)(Lipoyl/octanoyl transferase) (Octanoyl-[acyl-carrier-protein]-protein N-octanoyltransferase) A8K5M9Uncharacterized protein C15orf62, METWRKGSFRN (SEQ ID NO: mitochondrial255) A8MUP2 Methyltransferase-like protein 12,MAALRRMLHLPSLMMGTCRPFAGSLADS (SEQ ID NO: mitochondrial (EC 2.1.1.-) 256)O00142 Thymidine kinase 2, mitochondrial (ECMLLWPLRGWAARALRCFGPGSRGSPASG (SEQ ID NO: 2.7.1.21) (Mt-TK) PGPRR 257)O00217 NADH dehydrogenase [ubiquinone] iron-MRCLTTPMLLRALAQAARAGPPGGRSLH (SEQ ID NO:sulfur protein 8, mitochondrial (EC 1.6.5.3) SSAVAA 258)(EC 1.6.99.3) (Complex I-23kD) (CI-23kD)(NADH-ubiquinone oxidoreductase 23 kDa subunit) (TYKY subunit) O00330Pyruvate dehydrogenase protein X component, MAASWRLGCDPRLLRYLVGFPGRRSVGL(SEQ ID NO: mitochondrial (Dihydrolipoamide VKGALGWSVSRGANWRWFHSTQWLR259) dehydrogenase-binding protein of pyruvatedehydrogenase complex) (E3-binding protein)(E3BP) (Lipoyl-containing pyruvatedehydrogenase complex component X) (proX) O00411DNA-directed RNA polymerase, MSALCWGRGAAGLKRALRPCGRPGLPGK (SEQ ID NO:mitochondrial (MtRPOL) (EC 2.7.7.6) EGTAGGVCGPRRS 260) O00746Nucleoside diphosphate kinase, mitochondrialMGGLFWRSALRGLRCGPRAPGPSLLVRH (SEQ ID NO:(NDK) (NDP kinase, mitochondrial) (EC GSGGP 261)2.7.4.6) (Nucleoside diphosphate kinase D) (NDPKD) (nm23-H4) O14521Succinate dehydrogenase [ubiquinone] MAVLWRLSAVCGALGGRALLLRTPVVRP(SEQ ID NO: cytochrome b small subunit, mitochondrialAHISAFLQDRPIPEWCGVQHIHLSPSHH 262) (CybS) (CII-4) (QPs3) (Succinatedehydrogenase complex subunit D) (Succinate-ubiquinone oxidoreductase cytochrome b smallsubunit) (Succinate-ubiquinone reductase membrane anchor subunit) O14548Cytochrome c oxidase subunit 7A-related MYYKFSGFTQKLAGAWASEAYSPQGLKP(SEQ ID NO: protein, mitochondrial (COX7a-relatedVVSTEAPPIIFATPTKLTSDSTVYDYA 263)protein) (Cytochrome c oxidase subunit VIIa- related protein) (EB1) mMCMMouse methylmalonyl-CoA mutase MLRAKNQLFLLSPHYLKQLNIPSASRWK (SEQ ID RLNO: 264) hMCM Human methylmalonyl-CoA mutaseMLRAKNQLFLLSPHYLRQVKESSGSRLI (SEQ ID QQRL NO: 265)

In some embodiments, the nucleic acid sequence encoding a mitochondrialtransit peptide has at least about 70%, at least about 80%, at leastabout 90%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, at least about 99%, or about 100% sequence identityto a sequence selected from the group of nucleotides 1 to 96 of SEQ IDNOs: 1-207, 732-765, and 772, wherein the transit peptide is capable oftargeting or carrying the MCM polypeptide into the mitochondria.

In some embodiments, the nucleic acid sequence encoding a mitochondrialtransit peptide has at least about 70%, at least about 80%, at leastabout 90%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, at least about 99%, or about 100% sequence identityto a sequence in Table 1 (SEQ ID NOs: 251, 252, 253, 254, 255, 256, 257,258, 259, 260, 261, 262, 263, 264, or 265), wherein the transit peptideis capable of targeting or carrying the MCM polypeptide into themitochondria. In some embodiments, the nucleic acid sequence encoding amitochondrial transit peptide has at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, or about 100%sequence identity to a sequence selected from the group consisting ofSEQ ID NOs: 270 to 719, wherein the transit peptide is capable oftargeting or carrying the MCM polypeptide into the mitochondria.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 99% or 100% sequence identity tonucleotide 1 to nucleotide 2250 of SEQ ID NO: 734, wherein the ORFencodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 98%, at least 99%, or 100% sequenceidentity to nucleotide 1 to nucleotide 2250 of SEQ ID NO: 732, whereinthe ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to a sequence selected from the group consistingof nucleotides 1 to nucleotides 2250 of SEQ ID NO: 182 and 733, whereinthe ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to a sequence selected from thegroup consisting of nucleotides 1 to nucleotides 2250 of SEQ ID NOs:735, 741, 743, 744, 748, 758, 762, and 765, wherein the ORF encodes anMCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to a sequenceselected from the group consisting of nucleotides 1 to nucleotides 2250of SEQ ID NOs: 180, 181, 736, 738, 739, 740, 742, 746, 747, 749, 750,751, 752, 753, 754, 755, 757, 759, 760, 761, and 763, wherein the ORFencodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to asequence selected from the group consisting of nucleotides 1 tonucleotides 2250 of SEQ ID NO: 745, 756, and 764, wherein the ORFencodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to a sequence selected from the group consisting of nucleotides1 to nucleotides 2250 of SEQ ID NO: 154, 165, 171, 173, and 175, whereinthe ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to a sequence selected from the group consistingof nucleotides 1 to nucleotides 2250 of SEQ ID NO: 151, 152, 153, 163,166, 167, 168, 169, 170, 172, 177, 178, 179, 187, and 204, wherein theORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to a sequence selected from thegroup consisting of nucleotides 1 to nucleotides 2250 of SEQ ID NO: 156,157, 158, 159, 160, 162, 164, 174, 176, 195, and 737, wherein the ORFencodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to a sequenceselected from the group consisting of nucleotides 1 to nucleotides 2250of SEQ ID NOs: 155, 161, and 203, wherein the ORF encodes an MCMpolypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to a sequence selected from the group consistingof nucleotides 1 to nucleotides 2250 of SEQ ID NOs: 71 and 128, whereinthe ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to a sequence selected from thegroup consisting of nucleotides 1 to nucleotides 2250 of SEQ ID NOs: 4,6, 8, 9, 11, 19, 22, 23, 24, 32, 33, 37, 40, 44, 45, 47, 51, 61, 64, 65,66, 79, 84, 86, 90, 91, 92, 100, 101, 112, 115, 117, 126, 129, 135, 136,146, 148, 184, 190, and 191, wherein the ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to a sequenceselected from the group consisting of nucleotides 1 to nucleotides 2250of SEQ ID NOs: 2, 3, 5, 7, 10, 12, 13, 14, 15, 16, 18, 20, 21, 26, 27,28, 29, 31, 34, 36, 38, 39, 41, 42, 43, 46, 48, 49, 52, 53, 54, 55, 56,57, 58, 59, 60, 62, 68, 69, 70, 72, 73, 74, 76, 77, 80, 83, 85, 88, 95,96, 97, 98, 102, 104, 105, 106, 107, 108, 109, 110, 113, 114, 120, 121,122, 123, 124, 127, 131, 132, 133, 134, 137, 138, 139, 140, 141, 142,144, 145, 147, 149, 150, 186, 188, 189, 192, 193, 194, 196, 198, 199,200, 202, 205, 206, and 207, wherein the ORF encodes an MCM polypeptide.

In some embodiments, the disclosure is directed to a polynucleotidecomprising an ORF having at least 79%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to asequence selected from the group consisting of nucleotides 1 tonucleotides 2250 of SEQ ID NOs: 1, 17, 25, 30, 35, 50, 63, 67, 75, 78,81, 82, 87, 89, 93, 94, 99, 103, 111, 116, 118, 119, 125, 130, 143, 183,185, 197, and 201, wherein the ORF encodes an MCM polypeptide.

In some embodiments, the polynucleotide includes from about 1500 toabout 100,000 nucleotides (e.g., from about 1500 to 2500, from about1800 to about 2600, from about 1900 to about 2600, from about 2000 toabout 2700, from 2154 to 2,750, from 2154 to 3,000, from 2154 to 5,000,from 2154 to 7,000, from 2154 to 10,000, from 2154 to 25,000, from 2154to 50,000, from 2154 to 70,000, from 2154 to 100,000, from 2250 to 2750,from 2250 to 3,000, from 2250 to 5,000, from 2250 to 7,000, from 2250 to10,000, from 2250 to 25,000, from 2250 to 50,000, from 2250 to 70,000,and from 2250 to 100,000 nucleotides).

In some embodiments, the polynucleotides of the present disclosure canfurther comprise at least one nucleic acid sequence that is non-coding.

In some embodiments, the length of a region encoding at least onepolypeptide of interest is greater than about 2154 nucleotides in length(e.g., at least or greater than about 2154, 2,250, 2,500, 3,000, 4,000,4,100, 4,200, 4,300, 4,400, 4,500, 4,600, 4,700, 4,800, 4,900, 5,000,5,100, 5,200, 5,300, 5,400, 5,500, 5,600, 5,700, 5,800, 5,900, 6,000,7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000,70,000, 80,000, 90,000 or up to and including 100,000 nucleotides). Asused herein, such a region can be referred to as a “coding region” or“region encoding.”

In some embodiments, the polynucleotides of the present disclosure are,or function as, a messenger RNA (mRNA). As used herein, the term“messenger RNA” (mRNA) refers to any polynucleotide that encodes atleast one polypeptide of interest and that is capable of beingtranslated to produce the encoded polypeptide of interest in vitro, invivo, in situ or ex vivo. Exemplary mRNAs that can be used are listed inSEQ ID NOs: 776-771.

Optimized Polynucleotides Encoding MCM

The polynucleotides of the disclosure, their regions or parts orsubregions are sequence-optimized. Sequence optimization methods areknown in the art and can be useful to achieve one or more desiredresults. These results include to match codon frequencies in target andhost organisms to ensure proper folding, bias GC content to increasemRNA stability or reduce secondary structures, minimize tandem repeatcodons or base runs that can impair gene construction or expression,customize transcriptional and translational control regions, insert orremove protein trafficking sequences, remove/add post translationmodification sites in encoded protein (e.g., glycosylation sites), add,remove or shuffle protein domains, insert or delete restriction sites,modify ribosome binding sites and mRNA degradation sites, to adjusttranslational rates to allow the various domains of the protein to foldproperly, or to reduce or eliminate problem secondary structures withinthe polynucleotide. Sequence optimization tools, algorithms and servicesare known in the art, non-limiting examples include services fromGeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.) and/orproprietary methods. In some embodiments, the ORF sequence is optimizedusing optimization algorithms. Codon options for each amino acid aregiven in Table 2.

TABLE 2 Codon Options Single Letter Amino Acid Code Codon OptionsIsoleucine I ATT, ATC, ATA Leucine L CTT, CTC, CTA, CTG, TTA, TTG ValineV GTT, GTC, GTA, GTG Phenylalanine F TTT, TTC Methionine M ATG CysteineC TGT, TGC Alanine A GCT, GCC, GCA, GCG Glycine G GGT, GGC, GGA, GGGProline P CCT, CCC, CCA, CCG Threonine T ACT, ACC, ACA, ACG Serine STCT, TCC, TCA, TCG, AGT, AGC Tyrosine Y TAT, TAC Tryptophan W TGGGlutamine Q CAA, CAG Asparagine N AAT, AAC Histidine H CAT, CACGlutamic acid E GAA, GAG Aspartic acid D GAT, GAC Lysine K AAA, AAGArginine R CGT, CGC, CGA, CGG, AGA, AGG Selenocysteine SecUGA in mRNA in presence of Selenocysteine insertion element (SECTS)Stop codons Stop TAA, TAG, TGA

In some embodiments, the percentage of uracil or thymine nucleobases ina sequence-optimized nucleotide sequence (e.g., encoding an MCMpolypeptide, a functional fragment, or a variant thereof) is modified(e.g., reduced) with respect to the percentage of uracil or thyminenucleobases in the reference wild-type nucleotide sequence. Such asequence is referred to as a uracil-modified or thymine-modifiedsequence. The percentage of uracil or thymine content in a nucleotidesequence can be determined by dividing the number of uracils or thyminesin a sequence by the total number of nucleotides and multiplying by 100.In some embodiments, the sequence-optimized nucleotide sequence has alower uracil or thymine content than the uracil or thymine content inthe reference wild-type sequence. In some embodiments, the uracil orthymine content in a sequence-optimized nucleotide sequence of thedisclosure is greater than the uracil or thymine content in thereference wild-type sequence and still maintain beneficial effects,e.g., increased expression and/or reduced Toll-Like Receptor (TLR)response when compared to the reference wild-type sequence.

The uracil or thymine content of wild-type MCM is about 26.67%. In someembodiments, the uracil or thymine content of a uracil- orthymine-modified sequence encoding an MCM polypeptide is less than26.67%. In some embodiments, the uracil or thymine content of a uracil-or thymine-modified sequence encoding an MCM polypeptide of thedisclosure is less than 19%, less that 18%, less than 17%, less than16%, less than 15%, less than 14%, less than 13%, less than 12%, lessthan 11%, or less than 10%. In some embodiments, the uracil or thyminecontent is not less than 18%, 17%, 16%, 15%, 14%, 13%, 12%, or 11%. Theuracil or thymine content of a sequence disclosed herein, i.e., itstotal uracil or thymine content, is abbreviated herein as % U_(TL) or %T_(TL).

In some embodiments, the uracil or thymine content (% U_(TL) or %T_(TL)) of a uracil- or thymine-modified sequence encoding an MCMpolypeptide of the disclosure is between 11% and 26%, between 12% and25%, between 12% and 24%, between 13% and 23%, between 13% and 22%,between 14% and 21%, between 14% and 20%, between 14% and 19%, between14% and 18%, between 14% and 17%, or between 14% and 16%.

In some embodiments, the uracil or thymine content (% U_(TL) or %T_(TL)) of a uracil- or thymine-modified sequence encoding an MCMpolypeptide of the disclosure is between 13% and 17%, between 13% and16%, or between 14% and 16%.

In a particular embodiment, the uracil or thymine content (% U_(TL) or %T_(TL)) of a uracil- or thymine modified sequence encoding an MCMpolypeptide of the disclosure is between about 14% and about 16%, e.g.,between 14% and 15%.

A uracil- or thymine-modified sequence encoding an MCM polypeptide ofthe disclosure can also be described according to its uracil or thyminecontent relative to the uracil or thymine content in the correspondingwild-type nucleic acid sequence (% U_(WT) or % T_(WT)), or according toits uracil or thymine content relative to the theoretical minimum uracilor thymine content of a nucleic acid encoding the wild-type proteinsequence (% U_(TM) or (% T_(TM)).

The phrases “uracil or thymine content relative to the uracil or thyminecontent in the wild type nucleic acid sequence,” refers to a parameterdetermined by dividing the number of uracils or thymines in asequence-optimized nucleic acid by the total number of uracils orthymines in the corresponding wild-type nucleic acid sequence andmultiplying by 100. This parameter is abbreviated herein as % U_(WT) or% T_(WT).

In some embodiments, the % U_(WT) or % T_(WT) of a uracil- orthymine-modified sequence encoding an MCM polypeptide of the disclosureis above 50%, above 55%, above 60%, above 65%, above 70%, above 75%,above 80%, above 85%, above 90%, or above 95%.

In some embodiments, the % U_(WT) or % T_(WT) of a uracil- or thyminemodified sequence encoding an MCM polypeptide of the disclosure isbetween 42% and 68%, between 43% and 67%, between 44% and 66%, between45% and 65%, between 46% and 64%, between 47% and 63%, between 48% and62%, between 49% and 61%, between 50% and 60%, between 51% and 59%, orbetween 52% and 58%.

In some embodiments, the % U_(WT) or % T_(WT) of a uracil- orthymine-modified sequence encoding an MCM polypeptide of the disclosureis between 51% and 60%, between 51% and 59%, between 52% and 59%,between 52% and 58%, or between 53% and 58%.

In a particular embodiment, the % U_(WT) or % T_(WT) of a uracil- orthymine-modified sequence encoding an MCM polypeptide of the disclosureis between about 53% and about 58%.

Uracil- or thymine-content relative to the uracil or thymine theoreticalminimum, refers to a parameter determined by dividing the number ofuracils or thymines in a sequence-optimized nucleotide sequence by thetotal number of uracils or thymines in a hypothetical nucleotidesequence in which all the codons in the hypothetical sequence arereplaced with synonymous codons having the lowest possible uracil orthymine content and multiplying by 100. This parameter is abbreviatedherein as % U_(TM) or % T_(TM)

For DNA it is recognized that thymine is present instead of uracil, andone would substitute T where U appears. Thus, all the disclosuresrelated to, e.g., % U_(TM), % U_(WT), or % U_(TL), with respect to RNAare equally applicable to % T_(TM), % T_(WT), or % T_(TL) with respectto DNA.

In some embodiments, the % U_(TM) of a uracil-modified sequence encodingan MCM polypeptide of the disclosure is below 300%, below 295%, below290%, below 285%, below 280%, below 275%, below 270%, below 265%, below260%, below 255%, below 250%, below 245%, below 240%, below 235%, below230%, below 225%, below 220%, below 215%, below 200%, below 195%, below190%, below 185%, below 180%, below 175%, below 170%, below 165%, below160%, below 155%, below 150%, below 145%, below 140%, below 139%, below138%, below 137%, below 136%, below 135%, below 134%, below 133%, below132%, below 131%, below 130%, below 129%, below 128%, below 127%, below126%, below 125%, below 124%, below 123%, below 122%, below 121%, below120%, below 119%, below 118%, or below 117%.

In some embodiments, the % U_(TM) of a uracil-modified sequence encodingan MCM polypeptide of the disclosure is above 100%, above 101%, above102%, above 103%, above 104%, above 105%, above 106%, above 107%, above108%, above 109%, above 110%, above 111%, above 112%, above 113%, above114%, above 115%, above 116%, above 117%, above 118%, above 119%, above120%, above 121%, above 122%, above 123%, above 124%, above 125%, orabove 126%, above 127%, above 128%, or above 129%.

In some embodiments, the % U_(TM) of a uracil-modified sequence encodingan MCM polypeptide of the disclosure is between 123% and 125%, between122% and 126%, between 121% and 127%, between 120% and 128%, between119% and 129%, between 118% and 130%, between 117% and 131%, between116% and 132%, between 115% and 133%, between 114% and 134%, between113% and 135%, between 112% and 136%, or between 111% and 137%.

In some embodiments, the % U_(TM) of a uracil-modified sequence encodingan MCM polypeptide of the disclosure is between about 118% and about129%.

In some embodiments, a uracil-modified sequence encoding an MCMpolypeptide of the disclosure has a reduced number of consecutiveuracils with respect to the corresponding wild-type nucleic acidsequence. For example, two consecutive leucines can be encoded by thesequence CUUUUG, which includes a four uracil cluster. Such asubsequence can be substituted, e.g., with CUGCUC, which removes theuracil cluster.

Phenylalanine can be encoded by UUC or UUU. Thus, even if phenylalaninesencoded by UUU are replaced by UUC, the synonymous codon still containsa uracil pair (UU). Accordingly, the number of phenylalanines in asequence establishes a minimum number of uracil pairs (UU) that cannotbe eliminated without altering the number of phenylalanines in theencoded polypeptide. For example, if the polypeptide, e.g., wild typeMCM, has 27, 28, 29, or 30 phenylalanines, the absolute minimum numberof uracil pairs (UU) in that uracil-modified sequence encoding thepolypeptide, e.g., wild type MCM, can contain is 27, 28, 29, or 30,respectively.

Wild type MCM contains 82 uracil pairs (UU), and 29 uracil triplets(UUU). In some embodiments, a uracil-modified sequence encoding an MCMpolypeptide of the disclosure has a reduced number of uracil triplets(UUU) with respect to the wild-type nucleic acid sequence. In someembodiments, a uracil-modified sequence encoding an MCM polypeptide ofthe disclosure contains 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or no uraciltriplets (UUU).

In some embodiments, a uracil-modified sequence encoding an MCMpolypeptide has a reduced number of uracil pairs (UU) with respect tothe number of uracil pairs (UU) in the wild-type nucleic acid sequence.In some embodiments, a uracil-modified sequence encoding an MCMpolypeptide of the disclosure has a number of uracil pairs (UU)corresponding to the minimum possible number of uracil pairs (UU) in thewild-type nucleic acid sequence, e.g., 28 uracil pairs in the case ofwild type MCM.

In some embodiments, a uracil-modified sequence encoding an MCMpolypeptide of the disclosure has at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, or 54 uracil pairs (UU) less than thenumber of uracil pairs (UU) in the wild-type nucleic acid sequence. Insome embodiments, a uracil-modified sequence encoding an MCM polypeptideof the disclosure has between 20 and 35 uracil pairs (UU).

The phrase “uracil pairs (UU) relative to the uracil pairs (UU) in thewild type nucleic acid sequence,” refers to a parameter determined bydividing the number of uracil pairs (UU) in a sequence-optimizednucleotide sequence by the total number of uracil pairs (UU) in thecorresponding wild-type nucleotide sequence and multiplying by 100. Thisparameter is abbreviated herein as % UU_(wt).

In some embodiments, a uracil-modified sequence encoding an MCMpolypeptide of the disclosure has a % UU_(wt) less than 90%, less than85%, less than 80%, less than 75%, less than 70%, less than 65%, lessthan 60%, less than 65%, less than 60%, less than 55%, less than 50%,less than 40%, less than 30%, or less than 20%.

In some embodiments, a uracil-modified sequence encoding an MCMpolypeptide has a % UU_(wt) between 20% and 50%. In a particularembodiment, a uracil-modified sequence encoding an MCM polypeptide ofthe disclosure has a % UU_(wt) between 24% and 43%.

In some embodiments, the polynucleotide of the disclosure comprises auracil-modified sequence encoding an MCM polypeptide disclosed herein.In some embodiments, the uracil-modified sequence encoding an MCMpolypeptide comprises at least one chemically modified nucleobase, e.g.,5-methoxyuracil. In some embodiments, at least 95% of a nucleobase(e.g., uracil) in a uracil-modified sequence encoding an MCM polypeptideof the disclosure are modified nucleobases. In some embodiments, atleast 95% of uracil in a uracil-modified sequence encoding an MCMpolypeptide is 5-methoxyuracil.

In some embodiments, the “guanine content of the sequence optimized ORFencoding MCM with respect to the theoretical maximum guanine content ofa nucleotide sequence encoding the MCM polypeptide,” abbreviated as %G_(TMX) is at least 69%, at least 70%, at least 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, or about100%. In some embodiments, the % G_(TMX) is between about 70% and about80%, between about 71% and about 79%, between about 71% and about 78%,between about 71% and about 77% or between about 71% and about 76%.

In some embodiments, the “cytosine content of the ORF relative to thetheoretical maximum cytosine content of a nucleotide sequence encodingthe MCM polypeptide,” abbreviated as % C_(TMX), is at least about 68%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, or about 100%. Insome embodiments, the % C_(TMX) is between about 68% and about 77%,between about 69% and about 76%, or between about 70% and about 75%.

In some embodiments, the “guanine and cytosine content (G/C) of the ORFrelative to the theoretical maximum G/C content in a nucleotide sequenceencoding the MCM polypeptide,” abbreviated as % G/C_(TMX) is at leastabout 85%, at least about 90%, at least about 95%, or about 100%. The %G/C_(TMX) is between about 85% and about 100%, between about 89% andabout 96%, between about 90% and about 95%, or between about 91% andabout 94%.

In some embodiments, the “G/C content in the ORF relative to the G/Ccontent in the corresponding wild-type ORF,” abbreviated as % G/C_(WT)is at least 120%, at least 130%, at least 140%, at least 141%, at least142%, at least 143%, at least 144%, at least 145%, at least 146%, atleast 147%, at least 150%, or at least 155%.

In some embodiments, the average G/C content in the 3rd codon positionin the ORF is at least 50%, at least 51%, at least 52%, at least 53%, atleast 54%, at least 55%, at least 56%, at least 57%, at least 58%, atleast 59%, or at least 60% higher than the average G/C content in the3rd codon position in the corresponding wild-type ORF.

In some embodiments, the polynucleotide of the disclosure comprises anopen reading frame (ORF) encoding an MCM polypeptide, wherein the ORFhas been sequence optimized, and wherein each of % U_(TL), % U_(WT), %U_(TM), % G_(TL), % G_(WT), % G_(TMX), % C_(TL), % C_(WT), % C_(TMX), %G/C_(TL), % G/C_(WT), or % G/C_(TMX), alone or in a combination thereofis in a range between (i) a maximum corresponding to the parameter'smaximum value (MAX) plus about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 standard deviations (STDDEV), and (ii) a minimum corresponding to the parameter's minimum value(MIN) less 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5,8, 8.5, 9, 9.5, or 10 standard deviations (STD DEV).

Features, which can be considered beneficial in some embodiments of thepresent disclosure, can be encoded by regions of the polynucleotide andsuch regions can be upstream (5′) or downstream (3′) to, or within, aregion that encodes a polypeptide. These regions can be incorporatedinto the polynucleotide before and/or after sequence optimization of theprotein encoding region or open reading frame (ORF). It is not requiredthat a polynucleotide contain both a 5′ and 3′ flanking region. Examplesof such features include, but are not limited to, untranslated regions(UTRs), Kozak sequences, an oligo(dT) sequence, and detectable tags andcan include multiple cloning sites that can have XbaI recognition.

In some embodiments, a 5′ UTR and/or a 3′ UTR region can be provided asflanking regions. Multiple 5′ or 3′ UTRs can be included in the flankingregions and can be the same or of different sequences. Any portion ofthe flanking regions, including none, can be sequence-optimized and anycan independently contain one or more different structural or chemicalmodifications, before and/or after sequence optimization.

In some embodiments, the polynucleotide of the disclosure comprises,consists essentially or, or consists of the sequence set forth as SEQ IDNO: 769, wherein thymidine is changed to uridine. In other embodiments,the polynucleotide of the disclosure comprises, consists essentially or,or consists of the sequence set forth as SEQ ID NO: 770, whereinthymidine is changed to uridine. In other embodiments, thepolynucleotide does not comprise a polyC.

After optimization (if desired), the polynucleotides components arereconstituted and transformed into a vector such as, but not limited to,plasmids, viruses, cosmids, and artificial chromosomes. For example, theoptimized polynucleotide can be reconstituted and transformed intochemically competent E. coli, yeast, neurospora, maize, drosophila, etc.where high copy plasmid-like or chromosome structures occur by methodsdescribed herein.

Synthetic polynucleotides and their nucleic acid analogs play animportant role in the research and studies of biochemical processes.Various enzyme-assisted and chemical-based methods have been developedto synthesize polynucleotides and nucleic acids.

Enzymatic methods include in vitro transcription that uses RNApolymerases to synthesize the polynucleotides of the present disclosure.Enzymatic methods and RNA polymerases for transcription are described inInternational Patent Application No. PCT/US2014/53907, the contents ofwhich are herein incorporated by reference in its entirety.

Solid-phase chemical synthesis can be used to manufacture thepolynucleotides described herein or portions thereof. Solid-phasechemical synthesis manufacturing of the polynucleotides described hereinare described in International Patent Application No. PCT/US2014/53907,the contents of which are herein incorporated by reference in itsentirety.

Liquid phase chemical synthesis can be used to manufacture thepolynucleotides described herein or portions thereof. Liquid phasechemical synthesis manufacturing of the polynucleotides described hereinare described in International Patent Application No. PCT/US2014/53907,the contents of which are herein incorporated by reference in itsentirety.

Combinations of different synthetic methods can be used to manufacturethe polynucleotides described herein or portions thereof. Thesecombinations are described in International Patent Application No.PCT/US2014/53907, the contents of which are herein incorporated byreference in its entirety.

Small region synthesis can be used for regions or subregions of thepolynucleotides of the present disclosure. These synthesis methods aredescribed in International Patent Application No. PCT/US2014/53907, thecontents of which are herein incorporated by reference in its entirety.

Ligation of polynucleotide regions or subregions can be used to preparethe polynucleotides described herein. These ligation methods aredescribed in International Patent Application No. PCT/US2014/53907, thecontents of which are herein incorporated by reference in its entirety.

Polypeptides Encoded by the Polynucleotides of the Disclosure

In some embodiments, the MCM polypeptides encoded by polynucleotides ofthe disclosure peptide are functional MCM. As used herein, the term“MCM” protein is used interchangeably with “MUT” protein. Therefore,human MCM protein can be written as human MUT or hMUT, and murine MCMprotein can be written as murine MUT or mMUT. In some embodiments, theMCM polypeptides encoded by polynucleotides of the disclosure peptideare variants, peptides or polypeptides containing substitutions,insertions and/or additions, deletions and covalent modifications withrespect to an MCM peptide sequence. For example, sequence tags or aminoacids, such as one or more lysines, can be added to the peptidesequences encoded by the polynucleotides of the disclosure (e.g., at theN-terminal or C-terminal ends). Sequence tags can be used for peptidepurification or localization. Lysines can be used to increase peptidesolubility or to allow for biotinylation. In some embodiments, aminoacid residues located at the carboxy and amino terminal regions of apolypeptide encoded by the polynucleotides of the disclosure canoptionally be deleted providing for truncated sequences.

In some embodiments, the polynucleotides described herein encode asubstitutional variant of an MCM protein. The substitutional variant cancomprise one, two, three or more than three substitutions.“Substitutional variants” when referring to polypeptides are those thathave at least one amino acid residue in a native or starting sequenceremoved and a different amino acid inserted in its place at the sameposition. The substitutions can be single, where only one amino acid inthe molecule has been substituted, or they can be multiple, where two ormore amino acids have been substituted in the same molecule.

In some embodiments, the polynucleotides described herein encode avariant of an MCM protein with one or more conservative amino acidssubstitutions. As used herein the term “conservative amino acidsubstitution” refers to the substitution of an amino acid that isnormally present in the sequence with a different amino acid of similarsize, charge, or polarity. Examples of conservative substitutionsinclude the substitution of a non-polar (hydrophobic) residue such asisoleucine, valine and leucine for another non-polar residue. Likewise,examples of conservative substitutions include the substitution of onepolar (hydrophilic) residue for another such as between arginine andlysine, between glutamine and asparagine, and between glycine andserine. Additionally, the substitution of a basic residue such aslysine, arginine or histidine for another, or the substitution of oneacidic residue such as aspartic acid or glutamic acid for another acidicresidue are additional examples of conservative substitutions. Examplesof non-conservative substitutions include the substitution of anon-polar (hydrophobic) amino acid residue such as isoleucine, valine,leucine, alanine, methionine for a polar (hydrophilic) residue such ascysteine, glutamine, glutamic acid or lysine and/or a polar residue fora non-polar residue.

In other embodiments, the polynucleotides encode an insertional MCMvariant. “Insertional variants” when referring to polypeptides are thosewith one or more amino acids inserted immediately adjacent to an aminoacid at a particular position in a native or starting sequence.“Immediately adjacent” to an amino acid means connected to either thealpha-carboxy or alpha-amino functional group of the amino acid.

In other embodiments, the polynucleotides of the disclosure encode adeletional MCM variant. “Deletional variants” when referring topolypeptides are those with one or more amino acids in the native orstarting amino acid sequence removed. Ordinarily, deletional variantswill have one or more amino acids deleted in a particular region of themolecule.

In some embodiments, the polynucleotides of the disclosure encode acovalent derivative. “Covalent derivatives” when referring topolypeptides include modifications of a native or starting protein withan organic proteinaceous or non-proteinaceous derivatizing agent, and/orpost-translational modifications. Covalent modifications aretraditionally introduced by reacting targeted amino acid residues of theprotein with an organic derivatizing agent that is capable of reactingwith selected side-chains or terminal residues, or by harnessingmechanisms of post-translational modifications that function in selectedrecombinant host cells. The resultant covalent derivatives are useful inprograms directed at identifying residues important for biologicalactivity, for immunoassays, or for the preparation of anti-proteinantibodies for immunoaffinity purification of the recombinantglycoprotein. Such modifications are within the ordinary skill in theart and are performed without undue experimentation.

Certain post-translational modifications are the result of the action ofrecombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues can be present in the polypeptides produced in accordancewith the present disclosure.

Other post-translational modifications include hydroxylation of prolineand lysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the alpha-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86(1983)).

“Features,” when referring to polypeptides, are defined as distinctamino acid sequence-based components of a molecule. Features of thepolypeptides encoded by the polynucleotides of the present disclosureinclude surface manifestations, local conformational shape, folds,loops, half-loops, domains, half-domains, sites, termini or anycombination thereof.

As used herein, when referring to polypeptides, the term “domain” refersto a motif of a polypeptide having one or more identifiable structuralor functional characteristics or properties (e.g., binding capacity,serving as a site for protein-protein interactions).

As used herein, when referring to polypeptides, the terms “site” as itpertains to amino acid based embodiments is used synonymously with“amino acid residue” and “amino acid side chain.” A site represents aposition within a peptide or polypeptide that can be modified,manipulated, altered, derivatized or varied within the polypeptide basedmolecules of the present disclosure.

As used herein the terms “termini” or “terminus,” when referring topolypeptides, refers to an extremity of a peptide or polypeptide. Suchextremity is not limited only to the first or final site of the peptideor polypeptide but can include additional amino acids in the terminalregions. The polypeptide based molecules of the present disclosure canbe characterized as having both an N-terminus (terminated by an aminoacid with a free amino group (NH2)) and a C-terminus (terminated by anamino acid with a free carboxyl group (COOH)). Proteins of thedisclosure are in some cases made up of multiple polypeptide chainsbrought together by disulfide bonds or by non-covalent forces(multimers, oligomers). These sorts of proteins will have multiple N-and C-termini. Alternatively, the termini of the polypeptides can bemodified such that they begin or end, as the case can be, with anon-polypeptide based moiety such as an organic conjugate.

Once any of the features have been identified or defined as a desiredcomponent of a polypeptide to be encoded by the polynucleotide of thedisclosure, any of several manipulations and/or modifications of thesefeatures can be performed by moving, swapping, inverting, deleting,randomizing or duplicating. Furthermore, it is understood thatmanipulation of features can result in the same outcome as amodification to the molecules of the disclosure. For example, amanipulation that involved deleting a domain would result in thealteration of the length of a molecule just as modification of a nucleicacid to encode less than a full length molecule would.

Modifications and manipulations can be accomplished by methods known inthe art such as, but not limited to, site directed mutagenesis or apriori incorporation during chemical synthesis. The resulting modifiedmolecules can then be tested for activity using in vitro or in vivoassays such as those described herein or any other suitable screeningassay known in the art.

According to the present disclosure, the polypeptides can comprise aconsensus sequence that is discovered through rounds of experimentation.As used herein a “consensus” sequence is a single sequence thatrepresents a collective population of sequences allowing for variabilityat one or more sites.

As recognized by those skilled in the art, protein fragments, functionalprotein domains, and homologous proteins are also considered to bewithin the scope of polypeptides of interest of this disclosure. Forexample, provided herein is any protein fragment (meaning a polypeptidesequence at least one amino acid residue shorter than a referencepolypeptide sequence but otherwise identical) of a reference protein 10,20, 30, 40, 50, 60, 70, 80, 90, 100 or greater than 100 amino acids inlength. In another example, any protein that includes a stretch of about20, about 30, about 40, about 50, or about 100 amino acids that areabout 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about95%, or about 100% identical to any of the sequences described hereincan be utilized in accordance with the disclosure.

In certain embodiments, a polypeptide encoded by the polynucleotide ofthe disclosure includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations asshown in any of the sequences provided or referenced herein.

In some embodiments, the encoded polypeptide variant has the same or asimilar activity as the reference polypeptide. Alternatively, thevariant has an altered activity (e.g., increased or decreased) relativeto a reference polypeptide. Generally, variants of a particularpolynucleotide or polypeptide of the disclosure will have at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity tothat particular reference polynucleotide or polypeptide as determined bysequence alignment programs and parameters described herein and known tothose skilled in the art. Such tools for alignment include those of theBLAST suite (Stephen F. Altschul, Thomas L. Madden, Alejandro A.Schäffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman(1997), “Gapped BLAST and PSI-BLAST: a new generation of proteindatabase search programs,” Nucleic Acids Res. 25:3389-3402.) Other toolsare described herein, specifically in the definition of “Identity.”

Default parameters in the BLAST algorithm include, for example, anexpect threshold of 10, Word size of 28, Match/Mismatch Scores 1, −2,Gap costs Linear. Any filter can be applied as well as a selection forspecies specific repeats, e.g., Homo sapiens.

According to the present disclosure, the protein is encoded by apolynucleotide that can comprise at least a first region of linkednucleosides encoding at least one polypeptide of interest. Somepolypeptides encoded by the polynucleotides of interest of the presentdisclosure are listed in Table 3 below. In particular, Table 3 showshuman MCM wild type and mutant amino acid sequences.

TABLE 3 MCM Polypeptides and Polynucleotides SEQ ID No Gene SequenceSEQ ID FunctionalMLRAKNQLFLLSPHYLRQVKESSGSRLIQQRLLHQQQPLHPEWAALAKKQLKGKNPED NO: 208 HumanLIWHTPEGISIKPLYSKRDTMDLPEELPGVKPFTRGPYPTMYTFRPWTIRQYAGFSTV MCMEESNKFYKDNIKAGQQGLSVAFDLATHRGYDSDNPRVRGDVGMAGVAIDTVEDTKILFDGIPLEKMSVSMTMNGAVIPVLANFIVTGEEQGVPKEKLTGTIQNDILKEFMVRNTYIFPPEPSMKIIADIFEYTAKHMPKFNSISISGYHMQEAGADAILELAYTLADGLEYSRTGLQAGLTIDEFAPRLSFFWGIGMNFYMEIAKMRAGRRLWAHLIEKMFQPKNSKSLLLRAHCQTSGWSLTEQDPYNNIVRTAIEAMAAVEGGIQSLHINSFDEALGLPTVKSARIARNTQIIIQEESGIPKVADPWGGSYMMECLTNDVYDAALKLINEIEEMGGMAKAVAEGIPKLRIEECAARRQARIDSGSEVIVGVNKYQLEKEDAVEVLAIDNTSVRNRQIEKLKKIKSSRDQALAERCLAALTECAASGDGNILALAVDASRARCTVGEITDALKKVFGEHKANDRMVSGAYRQEFGESKEITSAIKRVHKFMEREGRRPRLLVAKMGQDGHDRGAKVIATGFADLGFDVDIGPLFQTPREVAQQAVDADVHAVGISTLAAGHKTLVPELIKELNSLGRPDILVMCGGVIPPQDYEFLFEVGVSNVFGPGTRIPKAAVQVLDDIEKCLEKKQQSV a.a. 33 to MatureLHQQQPLHPEWAALAKKQLKGKNPEDLIWHTPEGISIKPLYSKRDTMDLPEELPGVKP 750 of humanFIRGPYPTMYTFRPWTIRQYAGFSTVEESNKFYKDNIKAGQQGLSVAFDLATHRGYDS SEQ ID MCMDNPRVRGDVGMAGVAIDTVEDTKILFDGIPLEKMSVSMTMNGAVIPVLANFIVTGEEQ NO: 208GVPKEKLTGTIQNDILKEFMVRNTYIFPPEPSMKIIADIFEYTAKHMPKFNSISISGYHMQEAGADAILELAYTLADGLEYSRTGLQAGLTIDEFAPRLSFFWGIGMNFYMEIAKMRAGRRLWAHLIEKMFQPKNSKSLLLRAHCQTSGWSLTEQDPYNNIVRTAIEAMAAVFGGIQSLHINSFDEALGLPTVKSARIARNTQIIIQEESGIPKVADPWGGSYMMECLTNDVYDAALKLINEIEEMGGMAKAVAEGIPKLRIEECAARRQARIDSGSEVIVGVNKYQLEKEDAVEVLAIDNTSVRNRQIEKLKKIKSSRDQALAERCLAALTECAASGDGNILALAVDASRARCTVGEITDALKKVFGEHKANDRMVSGAYRQEFGESKEITSAIKRVHKFMEREGRRPRLLVAKMGQDGHDRGAKVIATGFADLGFDVDIGPLFQTPREVAQQAVDADVHAVGVSTLAAGHKTLVPELIKELNSLGRPDILVMCGGVIPPQDYEFLFEVGVSNVFGPGTRIPKAAVQVLDDIEKCLEKKQQSV SEQ ID FunctionalMLRAKNQLFLLSPHYLRQVKESSGSRLIQQRLLHQQQPLHPEWAALAKKQLKGKNPED NO: 209 HumanLIWHTPEGISVKPLYSKRDTMDLPEELPGVKPFTRGPYPTMYTFRPWTIRQYAGFSTV MCMEESNKFYKDNIKAGQQGLSVAFDLATHRGYDSDNPRVRGDVGMAGVAIDTVEDTKILF I69VDGIPLEKMSVSMTMNGAVIPVLANFIVTGEEQGVPKEKLTGTIQNDILKEFMVRNTYIFPPEPSMKIIADIFEYTAKHMPKFNSISISGYHMQEAGADAILELAYTLADGLEYSRTGLQAGLTIDEFAPRLSFFWGIGMNFYMEIAKMRAGRRLWAHLIEKMFQPKNSKSLLLRAHCQTSGWSLTEQDPYNNIVRTAIEAMAAVEGGIQSLHINSFDEALGLPTVKSARIARNTQIIIQEESGIPKVADPWGGSYMMECLINDVYDAALKLINEIEEMGGMAKAVAEGIPKLRIEECAARRQARIDSGSEVIVGVNKYQLEKEDAVEVLAIDNTSVRNRQIEKLKKIKSSRDQALAERCLAALTECAASGDGNILALAVDASRARCTVGEITDALKKVFGEHKANDRMVSGAYRQEFGESKEITSAIKRVHKFMEREGRRPRLLVAKMGQDGHDRGAKVIATGFADLGFDVDIGPLFQTPREVAQQAVDADVHAVGISTLAAGHKTLVPELIKELNSLGRPDILVMCGGVIPPQDYEFLFEVGVSNVFGPGTRIPKAAVQVLDDIEKCLEKKQQSV SEQ ID FunctionalMLRAKNQLFLLSPHYLRQVKESSGSRLIQQRLLHQQQPLHPEWAALAKKQLKGKNPED NO: 210 HumanLIWHTPEGISIKPLYSKRDTMDLPEELPGVKPFTRGPYPTMYTFRPWTIRQYAGFSTV MCMEESNKFYKDNIKAGQQGLSVAFDLATHRGYDSDNPRVRGDVGMAGVAIDTVEDTKILF A499TDGIPLEKMSVSMTMNGAVIPVLANFIVTGEEQGVPKEKLTGTIQNDILKEFMVRNTYIFPPEPSMKIIADIFEYTAKHMPKFNSISISGYHMQEAGADAILELAYTLADGLEYSRTGLQAGLTIDEFAPRLSFFWGIGMNFYMEIAKMRAGRRLWAHLIEKMFQPKNSKSLLLRAHCQTSGWSLTEQDPYNNIVRTAIEAMAAVEGGIQSLHINSFDEALGLPTVKSARIARNTQIIIQEESGIPKVADPWGGSYMMECLINDVYDAALKLINEIEEMGGMAKAVAEGIPKLRIEECAARRQARIDSGSEVIVGVNKYQLEKEDTVEVLAIDNTSVRNRQIEKLKKIKSSRDQALAERCLAALTECAASGDGNILALAVDASRARCTVGEITDALKKVFGEHKANDRMVSGAYRQEFGESKEITSAIKRVHKFMEREGRRPRLLVAKMGQDGHDRGAKVIATGFADLGFDVDIGPLFQTPREVAQQAVDADVHAVGISTLAAGHKTLVPELIKELNSLGRPDILVMCGGVIPPQDYEFLFEVGVSNVFGPGTRIPKAAVQVLDDIEKCLEKKQQSV SEQ ID FunctionalMLRAKNQLFLLSPHYLRQVKESSGSRLIQQRLLHQQQPLHPEWAALAKKQLKGKNPED NO: 211 HumanLIWHTPEGISIKPLYSKRDTMDLPEELPGVKPFTRGPYPTMYTFRPWTIRQYAGFSTV MCMEESNKFYKDNIKAGQQGLSVAFDLATHRGYDSDNPRVRGDVGMAGVAIDTVEDTKILF R532HDGIPLEKMSVSMTMNGAVIPVLANFIVTGEEQGVPKEKLTGTIQNDILKEFMVRNTYIFPPEPSMKIIADIFEYTAKHMPKFNSISISGYHMQEAGADAILELAYTLADGLEYSRTGLQAGLTIDEFAPRLSFFWGIGMNFYMEIAKMRAGRRLWAHLIEKMFQPKNSKSLLLRAHCQTSGWSLTEQDPYNNIVRTAIEAMAAVEGGIQSLHINSFDEALGLPTVKSARIARNTQIIIQEESGIPKVADPWGGSYMMECLINDVYDAALKLINEIEEMGGMAKAVAEGIPKLRIEECAARRQARIDSGSEVIVGVNKYQLEKEDAVEVLAIDNTSVRNRQIEKLKKIKSSRDQALAEHCLAALTECAASGDGNILALAVDASRARCTVGEITDALKKVFGEHKANDRMVSGAYRQEFGESKEITSAIKRVHKFMEREGRRPRLLVAKMGQDGHDRGAKVIATGFADLGFDVDIGPLFQTPREVAQQAVDADVHAVGISTLAAGHKTLVPELIKELNSLGRPDILVMCGGVIPPQDYEFLFEVGVSNVFGPGTRIPKAAVQVLDDIEKCLEKKQQSV SEQ ID FunctionalMLRAKNQLFLLSPHYLRQVKESSGSRLIQQRLLHQQQPLHPEWAALAKKQLKGKNPED NO: 212 HumanLIWHTPEGISIKPLYSKRDTMDLPEELPGVKPFTRGPYPTMYTFRPWTIRQYAGFSTV MCMEESNKFYKDNIKAGQQGLSVAFDLATHRGYDSDNPRVRGDVGMAGVAIDTVEDTKILF T598ADGIPLEKMSVSMTMNGAVIPVLANFIVTGEEQGVPKEKLTGTIQNDILKEFMVRNTYIFPPEPSMKIIADIFEYTAKHMPKENSISISGYHMQEAGADAILELAYTLADGLEYSRTGLQAGLTIDEFAPRLSFFWGIGMNFYMEIAKMRAGRRLWAHLIEKMFQPKNSKSLLLRAHCQTSGWSLTEQDPYNNIVRTAIEAMAAVEGGTQSLHINSFDEALGLPTVKSARIARNTQIIIQEESGIPKVADPWGGSYMMECLTNDVYDAALKLINEIEEMGGMAKAVAEGIPKLRIEECAARRQARIDSGSEVIVGVNKYQLEKEDAVEVLAIDNTSVRNRQIEKLKKIKSSRDQALAERCLAALTECAASGDGNILALAVDASRARCTVGEITDALKKVFGEHKANDRMVSGAYRQEFGESKEIASAIKRVHKFMEREGRRPRLLVAKMGQDGHDRGAKVIATGEADLGFDVDIGPLFQTPREVAQQAVDADVHAVGISTLAAGHKTLVPELIKELNSLGRPDILVMCGGVIPPQDYEFLFEVGVSNVFGPGTRIPKAAVQVLDDIEKCLEKKQQSV SEQ ID FunctionalMLRAKNQLFLLSPHYLRQVKESSGSRLIQQRLLHQQQPLHPEWAALAKKQLKGKNPED NO: 213 HumanLIWHTPEGISIKPLYSKRDTMDLPEELPGVKPFTRGPYPTMYTFRPWTIRQYAGFSTV MCMEESNKFYKDNIKAGQQGLSVAFDLATHRGYDSDNPRVRGDVGMAGVAIDTVEDTKILF I671VDGIPLEKMSVSMTMNGAVIPVLANFIVTGEEQGVPKEKLTGTIQNDILKEFMVRNTYIFPPEPSMKIIADIFEYTAKHMPKENSISISGYHMQEAGADAILELAYTLADGLEYSRTGLQAGLTIDEFAPRLSFFWGIGMNFYMEIAKMRAGRRLWAHLIEKMFQPKNSKSLLLRAHCQTSGWSLTEQDPYNNIVRTAIEAMAAVEGGTQSLHINSFDEALGLPTVKSARIARNTQIIIQEESGIPKVADPWGGSYMMECLTNDVYDAALKLINEIEEMGGMAKAVAEGIPKLRIEECAARRQARIDSGSEVIVGVNKYQLEKEDAVEVLAIDNTSVRNRQIEKLKKIKSSRDQALAERCLAALTECAASGDGNILALAVDASRARCTVGEITDALKKVFGEHKANDRMVSGAYRQEFGESKEITSAIKRVHKFMEREGRRPRLLVAKMGQDGHDRGAKVIATGEADLGFDVDIGPLFQTPREVAQQAVDADVHAVGVSTLAAGHKTLVPELIKELNSLGRPDILVMCGGVIPPQDYEFLFEVGVSNVFGPGTRIPKAAVQVLDDIEKCLEKKQQSV

II. MODIFIED POLYNUCLEOTIDES

The disclosure also includes a modified polynucleotide comprising thepolynucleotide described herein, i.e., a polynucleotide comprising anORF encoding an MCM polypeptide that contains polynucleotides that arechemically and/or structurally modified. When the polynucleotides of thepresent disclosure are chemically and/or structurally modified thepolynucleotides can be referred to as “modified polynucleotides.”

The present disclosure provides for modified nucleosides and nucleotidesof a polynucleotide (e.g., RNA polynucleotides, such as mRNApolynucleotides). A “nucleoside” refers to a compound containing a sugarmolecule (e.g., a pentose or ribose) or a derivative thereof incombination with an organic base (e.g., a purine or pyrimidine) or aderivative thereof (also referred to herein as “nucleobase”). Anucleotide” refers to a nucleoside, including a phosphate group.Modified nucleotides can by synthesized by any useful method, such as,for example, chemically, enzymatically, or recombinantly, to include oneor more modified or non-natural nucleosides. Polynucleotides cancomprise a region or regions of linked nucleosides. Such regions canhave variable backbone linkages. The linkages can be standardphosphodioester linkages, in which case the polynucleotides wouldcomprise regions of nucleotides.

The modifications can be various distinct modifications. In someembodiments, the regions can contain one, two, or more (optionallydifferent) nucleoside or nucleotide modifications. In some embodiments,a modified polynucleotide, introduced to a cell can exhibit reduceddegradation in the cell, as compared to an unmodified polynucleotide.

Structural Modifications

In some embodiments, the polynucleotides of the present disclosure arestructurally modified. As used herein, a “structural” modification isone in which two or more linked nucleosides are inserted, deleted,duplicated, inverted or randomized in a polynucleotide withoutsignificant chemical modification to the nucleotides themselves. Becausechemical bonds will necessarily be broken and reformed to effect astructural modification, structural modifications are of a chemicalnature and hence are chemical modifications. However, structuralmodifications will result in a different sequence of nucleotides. Forexample, the polynucleotide “ATCG” can be chemically modified to“AT-5meC-G”. The same polynucleotide can be structurally modified from“ATCG” to “ATCCCG”. Here, the dinucleotide “CC” has been inserted,resulting in a structural modification to the polynucleotide.

Chemical Modifications

In some embodiments, the polynucleotides of the present disclosure arechemically modified. As used herein in reference to a polynucleotide,the terms “chemical modification” or, as appropriate, “chemicallymodified” refer to modification with respect to adenosine (A), guanosine(G), uridine (U), thymidine (T) or cytidine (C) ribo- ordeoxyribonucleosides in one or more of their position, pattern, percentor population. Generally, herein, these terms are not intended to referto the ribonucleotide modifications in naturally occurring 5′-terminalmRNA cap moieties.

In some embodiments, the polynucleotides of the present disclosure canhave a uniform chemical modification of all or any of the samenucleoside type or a population of modifications produced by meredownward titration of the same starting modification in all or any ofthe same nucleoside type, or a measured percent of a chemicalmodification of all any of the same nucleoside type but with randomincorporation, such as where all uridines are replaced by a uridineanalog, e.g., pseudouridine or 5-methoxyuridine. In another embodiment,the polynucleotides can have a uniform chemical modification of two,three, or four of the same nucleoside type throughout the entirepolynucleotide (such as all uridines and all cytosines, etc. aremodified in the same way).

Modified nucleotide base pairing encompasses not only the standardadenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs,but also base pairs formed between nucleotides and/or modifiednucleotides comprising non-standard or modified bases, wherein thearrangement of hydrogen bond donors and hydrogen bond acceptors permitshydrogen bonding between a non-standard base and a standard base orbetween two complementary non-standard base structures. One example ofsuch non-standard base pairing is the base pairing between the modifiednucleotide inosine and adenine, cytosine or uracil. Any combination ofbase/sugar or linker can be incorporated into polynucleotides of thepresent disclosure.

The skilled artisan will appreciate that, except where otherwise noted,polynucleotide sequences set forth in the instant application willrecite “T”s in a representative DNA sequence but where the sequencerepresents RNA, the “T”s would be substituted for “U”s.

Modifications of polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) that are useful in the compositions, methods andsynthetic processes of the present disclosure include, but are notlimited to the following nucleotides, nucleosides, and nucleobases:2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine;2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonylcarbamoyladenosine; N6-glycinylcarbamoyladenosine;N6-isopentenyladenosine; N6-methyladenosine;N6-threonylcarbamoyladenosine; 1,2′-O-dimethyladenosine;1-methyladenosine; 2′-O-methyladenosine; 2′-O-ribosyladenosine(phosphate); 2-methyladenosine; 2-methylthio-N6 isopentenyladenosine;2-methylthio-N6-hydroxynorvalyl carbamoyladenosine;2′-O-methyladenosine; 2′-O-ribosyladenosine (phosphate);Isopentenyladenosine; N6-(cis-hydroxyisopentenyl)adenosine;N6,2′-O-dimethyladenosine; N6,2′-O-dimethyladenosine;N6,N6,2′-O-trimethyladenosine; N6,N6-dimethyladenosine;N6-acetyladenosine; N6-hydroxynorvalylcarbamoyladenosine;N6-methyl-N6-threonylcarbamoyladenosine; 2-methyladenosine;2-methylthio-N6-isopentenyladenosine; 7-deaza-adenosine;N1-methyl-adenosine; N6, N6 (dimethyl)adenine;N6-cis-hydroxy-isopentenyl-adenosine; α-thio-adenosine; 2(amino)adenine; 2 (aminopropyl)adenine; 2 (methylthio) N6(isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine;2-(aminopropyl)adenine; 2-(halo)adenine; 2-(halo)adenine;2-(propyl)adenine; 2′-Amino-2′-deoxy-ATP; 2′-Azido-2′-deoxy-ATP;2′-Deoxy-2′-a-aminoadenosine TP; 2′-Deoxy-2′-a-azidoadenosine TP; 6(alkyl)adenine; 6 (methyl)adenine; 6-(alkyl)adenine; 6-(methyl)adenine;7 (deaza)adenine; 8 (alkenyl)adenine; 8 (alkynyl)adenine; 8(amino)adenine; 8 (thioalkyl)adenine; 8-(alkenyl)adenine;8-(alkyl)adenine; 8-(alkynyl)adenine; 8-(amino)adenine; 8-(halo)adenine;8-(hydroxyl)adenine; 8-(thioalkyl)adenine; 8-(thiol)adenine;8-azido-adenosine; aza adenine; deaza adenine; N6 (methyl)adenine;N6-(isopentyl)adenine; 7-deaza-8-aza-adenosine; 7-methyladenine;1-Deazaadenosine TP; 2′Fluoro-N6-Bz-deoxyadenosine TP;2′-OMe-2-Amino-ATP; 2′O-methyl-N6-Bz-deoxyadenosine TP;2′-a-Ethynyladenosine TP; 2-aminoadenine; 2-Aminoadenosine TP;2-Amino-ATP; 2′-a-Trifluoromethyladenosine TP; 2-Azidoadenosine TP;2′-b-Ethynyladenosine TP; 2-Bromoadenosine TP;2′-b-Trifluoromethyladenosine TP; 2-Chloroadenosine TP;2′-Deoxy-2′,2′-difluoroadenosine TP; 2′-Deoxy-2′-a-mercaptoadenosine TP;2′-Deoxy-2′-a-thiomethoxyadenosine TP; 2′-Deoxy-2′-b-aminoadenosine TP;2′-Deoxy-2′-b-azidoadenosine TP; 2′-Deoxy-2′-b-bromoadenosine TP;2′-Deoxy-2′-b-chloroadenosine TP; 2′-Deoxy-2′-b-fluoroadenosine TP;2′-Deoxy-2′-b-iodoadenosine TP; 2′-Deoxy-2′-b-mercaptoadenosine TP;2′-Deoxy-2′-b-thiomethoxyadenosine TP; 2-Fluoroadenosine TP;2-Iodoadenosine TP; 2-Mercaptoadenosine TP; 2-methoxy-adenine;2-methylthio-adenine; 2-Trifluoromethyladenosine TP;3-Deaza-3-bromoadenosine TP; 3-Deaza-3-chloroadenosine TP;3-Deaza-3-fluoroadenosine TP; 3-Deaza-3-iodoadenosine TP;3-Deazaadenosine TP; 4′-Azidoadenosine TP; 4′-Carbocyclic adenosine TP;4′-Ethynyladenosine TP; 5′-Homo-adenosine TP; 8-Aza-ATP;8-bromo-adenosine TP; 8-Trifluoromethyladenosine TP; 9-DeazaadenosineTP; 2-aminopurine; 7-deaza-2,6-diaminopurine;7-deaza-8-aza-2,6-diaminopurine; 7-deaza-8-aza-2-aminopurine;2,6-diaminopurine; 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine;2-thiocytidine; 3-methylcytidine; 5-formylcytidine;5-hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine;2′-O-methylcytidine; 2′-O-methylcytidine; 5,2′-O-dimethylcytidine;5-formyl-2′-O-methylcytidine; Lysidine; N4,2′-O-dimethylcytidine;N4-acetyl-2′-O-methylcytidine; N4-methylcytidine;N4,N4-Dimethyl-2′-OMe-Cytidine TP; 4-methylcytidine; 5-aza-cytidine;Pseudo-iso-cytidine; pyrrolo-cytidine; α-thio-cytidine;2-(thio)cytosine; 2′-Amino-2′-deoxy-CTP; 2′-Azido-2′-deoxy-CTP;2′-Deoxy-2′-a-aminocytidine TP; 2′-Deoxy-2′-a-azidocytidine TP; 3(deaza) 5 (aza)cytosine; 3 (methyl)cytosine; 3-(alkyl)cytosine;3-(deaza) 5 (aza)cytosine; 3-(methyl)cytidine; 4,2′-O-dimethylcytidine;5 (halo)cytosine; 5 (methyl)cytosine; 5 (propynyl)cytosine; 5(trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine;5-(halo)cytosine; 5-(propynyl)cytosine; 5-(trifluoromethyl)cytosine;5-bromo-cytidine; 5-iodo-cytidine; 5-propynyl cytosine; 6-(azo)cytosine;6-aza-cytidine; aza cytosine; deaza cytosine; N4 (acetyl)cytosine;1-methyl-1-deaza-pseudoisocytidine; 1-methyl-pseudoisocytidine;2-methoxy-5-methyl-cytidine; 2-methoxy-cytidine;2-thio-5-methyl-cytidine; 4-methoxy-1-methyl-pseudoisocytidine;4-methoxy-pseudoisocytidine; 4-thio-1-methyl-1-deaza-pseudoisocytidine;4-thio-1-methyl-pseudoisocytidine; 4-thio-pseudoisocytidine;5-aza-zebularine; 5-methyl-zebularine; pyrrolo-pseudoisocytidine;Zebularine; (E)-5-(2-Bromo-vinyl)cytidine TP; 2,2′-anhydro-cytidine TPhydrochloride; 2′Fluor-N4-Bz-cytidine TP; 2′Fluoro-N4-Acetyl-cytidineTP; 2′-O-Methyl-N4-Acetyl-cytidine TP; 2′O-methyl-N4-Bz-cytidine TP;2′-a-Ethynylcytidine TP; 2′-a-Trifluoromethylcytidine TP;2′-b-Ethynylcytidine TP; 2′-b-Trifluoromethylcytidine TP;2′-Deoxy-2′,2′-difluorocytidine TP; 2′-Deoxy-2′-a-mercaptocytidine TP;2′-Deoxy-2′-a-thiomethoxycytidine TP; 2′-Deoxy-2′-b-aminocytidine TP;2′-Deoxy-2′-b-azidocytidine TP; 2′-Deoxy-2′-b-bromocytidine TP;2′-Deoxy-2′-b-chlorocytidine TP; 2′-Deoxy-2′-b-fluorocytidine TP;2′-Deoxy-2′-b-iodocytidine TP; 2′-Deoxy-2′-b-mercaptocytidine TP;2′-Deoxy-2′-b-thiomethoxycytidine TP; 2′-O-Methyl-5-(1-propynyl)cytidineTP; 3′-Ethynylcytidine TP; 4′-Azidocytidine TP; 4′-Carbocyclic cytidineTP; 4′-Ethynylcytidine TP; 5-(1-Propynyl)ara-cytidine TP;5-(2-Chloro-phenyl)-2-thiocytidine TP; 5-(4-Amino-phenyl)-2-thiocytidineTP; 5-Aminoallyl-CTP; 5-Cyanocytidine TP; 5-Ethynylara-cytidine TP;5-Ethynylcytidine TP; 5′-Homo-cytidine TP; 5-Methoxycytidine TP;5-Trifluoromethyl-Cytidine TP; N4-Amino-cytidine TP; N4-Benzoyl-cytidineTP; Pseudoisocytidine; 7-methylguanosine; N2,2′-O-dimethylguanosine;N2-methylguanosine; Wyosine; 1,2′-O-dimethylguanosine;1-methylguanosine; 2′-O-methylguanosine; 2′-O-ribosylguanosine(phosphate); 2′-O-methylguanosine; 2′-O-ribosylguanosine (phosphate);7-aminomethyl-7-deazaguanosine; 7-cyano-7-deazaguanosine; Archaeosine;Methylwyosine; N2,7-dimethylguanosine; N2,N2,2′-O-trimethylguanosine;N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine;N2,7,2′-O-trimethylguanosine; 6-thio-guanosine; 7-deaza-guanosine;8-oxo-guanosine; N1-methyl-guanosine; α-thio-guanosine; 2(propyl)guanine; 2-(alkyl)guanine; 2′-Amino-2′-deoxy-GTP;2′-Azido-2′-deoxy-GTP; 2′-Deoxy-2′-a-aminoguanosine TP;2′-Deoxy-2′-a-azidoguanosine TP; 6 (methyl)guanine; 6-(alkyl)guanine;6-(methyl)guanine; 6-methyl-guanosine; 7 (alkyl)guanine; 7(deaza)guanine; 7 (methyl)guanine; 7-(alkyl)guanine; 7-(deaza)guanine;7-(methyl)guanine; 8 (alkyl)guanine; 8 (alkynyl)guanine; 8(halo)guanine; 8 (thioalkyl)guanine; 8-(alkenyl)guanine;8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine;8-(hydroxyl)guanine; 8-(thioalkyl)guanine; 8-(thiol)guanine; azaguanine; deaza guanine; N (methyl)guanine; N-(methyl)guanine;1-methyl-6-thio-guanosine; 6-methoxy-guanosine;6-thio-7-deaza-8-aza-guanosine; 6-thio-7-deaza-guanosine;6-thio-7-methyl-guanosine; 7-deaza-8-aza-guanosine;7-methyl-8-oxo-guanosine; N2,N2-dimethyl-6-thio-guanosine;N2-methyl-6-thio-guanosine; 1-Me-GTP; 2′Fluoro-N2-isobutyl-guanosine TP;2′O-methyl-N2-isobutyl-guanosine TP; 2′-a-Ethynylguanosine TP;2′-a-Trifluoromethylguanosine TP; 2′-b-Ethynylguanosine TP;2′-b-Trifluoromethylguanosine TP; 2′-Deoxy-2′,2′-difluoroguanosine TP;2′-Deoxy-2′-a-mercaptoguanosine TP; 2′-Deoxy-2′-a-thiomethoxyguanosineTP; 2′-Deoxy-2′-b-aminoguanosine TP; 2′-Deoxy-2′-b-azidoguanosine TP;2′-Deoxy-2′-b-bromoguanosine TP; 2′-Deoxy-2′-b-chloroguanosine TP;2′-Deoxy-2′-b-fluoroguanosine TP; 2′-Deoxy-2′-b-iodoguanosine TP;2′-Deoxy-2′-b-mercaptoguanosine TP; 2′-Deoxy-2′-b-thiomethoxyguanosineTP; 4′-Azidoguanosine TP; 4′-Carbocyclic guanosine TP;4′-Ethynylguanosine TP; 5′-Homo-guanosine TP; 8-bromo-guanosine TP;9-Deazaguanosine TP; N2-isobutyl-guanosine TP; 1-methylinosine; Inosine;1,2′-O-dimethylinosine; 2′-O-methylinosine; 7-methylinosine;2′-O-methylinosine; Epoxyqueuosine; galactosyl-queuosine;Mannosylqueuosine; Queuosine; allyamino-thymidine; aza thymidine; deazathymidine; deoxy-thymidine; 2′-O-methyluridine; 2-thiouridine;3-methyluridine; 5-carboxymethyluridine; 5-hydroxyuridine;5-methyluridine; 5-taurinomethyl-2-thiouridine; 5-taurinomethyluridine;Dihydrouridine; Pseudouridine; (3-(3-amino-3-carboxypropyl)uridine;1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine;1-methylpseduouridine; 1-ethyl-pseudouridine; 2′-O-methyluridine;2′-O-methylpseudouridine; 2′-O-methyluridine; 2-thio-2′-O-methyluridine;3-(3-amino-3-carboxypropyl)uridine; 3,2′-O-dimethyluridine;3-Methyl-pseudo-Uridine TP; 4-thiouridine;5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl)uridine methylester; 5,2′-O-dimethyluridine; 5,6-dihydro-uridine;5-aminomethyl-2-thiouridine; 5-carbamoylmethyl-2′-O-methyluridine;5-carbamoylmethyluridine; 5-carboxyhydroxymethyluridine;5-carboxyhydroxymethyluridine methyl ester;5-carboxymethylaminomethyl-2′-O-methyluridine;5-carboxymethylaminomethyl-2-thiouridine;5-carboxymethylaminomethyl-2-thiouridine;5-carboxymethylaminomethyluridine; 5-carboxymethylaminomethyluridine;5-Carbamoylmethyluridine TP; 5-methoxycarbonylmethyl-2′-O-methyluridine;5-methoxycarbonylmethyl-2-thiouridine; 5-methoxycarbonylmethyluridine;5-methyluridine), 5-methoxyuridine; 5-methyl-2-thiouridine;5-methylaminomethyl-2-selenouridine; 5-methylaminomethyl-2-thiouridine;5-methylaminomethyluridine; 5-Methyldihydrouridine; 5-Oxyaceticacid-Uridine TP; 5-Oxyacetic acid-methyl ester-Uridine TP;N1-methyl-pseudo-uracil; N1-ethyl-pseudo-uracil; uridine 5-oxyaceticacid; uridine 5-oxyacetic acid methyl ester;3-(3-Amino-3-carboxypropyl)-Uridine TP;5-(iso-Pentenylaminomethyl)-2-thiouridine TP;5-(iso-Pentenylaminomethyl)-2′-O-methyluridine TP;5-(iso-Pentenylaminomethyl)uridine TP; 5-propynyl uracil;α-thio-uridine; 1(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-pseudouracil; 1(aminocarbonylethylenyl)-2(thio)-pseudouracil; 1(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1(aminocarbonylethylenyl)-4 (thio)pseudouracil; 1(aminocarbonylethylenyl)-pseudouracil; 1 substituted2(thio)-pseudouracil; 1 substituted 2,4-(dithio)pseudouracil; 1substituted 4 (thio)pseudouracil; 1 substituted pseudouracil;1-(aminoalkylamino-carbonylethylenyl)-2-(thio)-pseudouracil;1-Methyl-3-(3-amino-3-carboxypropyl) pseudouridine TP;1-Methyl-3-(3-amino-3-carboxypropyl)pseudo-UTP; 1-Methyl-pseudo-UTP;1-Ethyl-pseudo-UTP; 2 (thio)pseudouracil; 2′ deoxy uridine; 2′fluorouridine; 2-(thio)uracil; 2,4-(dithio)pseudouracil; 2′ methyl,2′amino, 2′azido, 2′fluro-guanosine; 2′-Amino-2′-deoxy-UTP;2′-Azido-2′-deoxy-UTP; 2′-Azido-deoxyuridine TP;2′-O-methylpseudouridine; 2′ deoxy uridine; 2′ fluorouridine;2′-Deoxy-2′-a-aminouridine TP; 2′-Deoxy-2′-a-azidouridine TP;2-methylpseudouridine; 3 (3 amino-3 carboxypropyl)uracil; 4(thio)pseudouracil; 4-(thio)pseudouracil; 4-(thio)uracil; 4-thiouracil;5 (1,3-diazole-1-alkyl)uracil; 5 (2-aminopropyl)uracil; 5(aminoalkyl)uracil; 5 (dimethylaminoalkyl)uracil; 5(guanidiniumalkyl)uracil; 5 (methoxycarbonylmethyl)-2-(thio)uracil; 5(methoxycarbonyl-methyl)uracil; 5 (methyl) 2 (thio)uracil; 5 (methyl)2,4 (dithio)uracil; 5 (methyl) 4 (thio)uracil; 5 (methylaminomethyl)-2(thio)uracil; 5 (methylaminomethyl)-2,4 (dithio)uracil; 5(methylaminomethyl)-4 (thio)uracil; 5 (propynyl)uracil; 5(trifluoromethyl)uracil; 5-(2-aminopropyl)uracil;5-(alkyl)-2-(thio)pseudouracil; 5-(alkyl)-2,4 (dithio)pseudouracil;5-(alkyl)-4 (thio)pseudouracil; 5-(alkyl)pseudouracil; 5-(alkyl)uracil;5-(alkynyl)uracil; 5-(allylamino)uracil; 5-(cyanoalkyl)uracil;5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil;5-(guanidiniumalkyl)uracil; 5-(halo)uracil;5-(1,3-diazole-1-alkyl)uracil; 5-(methoxy)uracil;5-(methoxycarbonylmethyl)-2-(thio)uracil;5-(methoxycarbonyl-methyl)uracil; 5-(methyl) 2(thio)uracil; 5-(methyl)2,4 (dithio)uracil; 5-(methyl) 4 (thio)uracil;5-(methyl)-2-(thio)pseudouracil; 5-(methyl)-2,4 (dithio)pseudouracil;5-(methyl)-4 (thio)pseudouracil; 5-(methyl)pseudouracil;5-(methylaminomethyl)-2 (thio)uracil;5-(methylaminomethyl)-2,4(dithio)uracil;5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil;5-(trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine;5-iodo-uridine; 5-uracil; 6 (azo)uracil; 6-(azo)uracil; 6-aza-uridine;allyamino-uracil; aza uracil; deaza uracil; N3 (methyl)uracil;Pseudo-UTP-1-2-ethanoic acid; Pseudouracil; 4-Thio-pseudo-UTP;1-carboxymethyl-pseudouridine; 1-methyl-1-deaza-pseudouridine;1-propynyl-uridine; 1-taurinomethyl-1-methyl-uridine;1-taurinomethyl-4-thio-uridine; 1-taurinomethyl-pseudouridine;2-methoxy-4-thio-pseudouridine; 2-thio-1-methyl-1-deaza-pseudouridine;2-thio-1-methyl-pseudouridine; 2-thio-5-aza-uridine;2-thio-dihydropseudouridine; 2-thio-dihydrouridine;2-thio-pseudouridine; 4-methoxy-2-thio-pseudouridine;4-methoxy-pseudouridine; 4-thio-1-methyl-pseudouridine;4-thio-pseudouridine; 5-aza-uridine; Dihydropseudouridine;(±)1-(2-Hydroxypropyl)pseudouridine TP;(2R)-1-(2-Hydroxypropyl)pseudouridine TP;(2S)-1-(2-Hydroxypropyl)pseudouridine TP;(E)-5-(2-Bromo-vinyl)ara-uridine TP; (E)-5-(2-Bromo-vinyl)uridine TP;(Z)-5-(2-Bromo-vinyl)ara-uridine TP; (Z)-5-(2-Bromo-vinyl)uridine TP;1-(2,2,2-Trifluoroethyl)-pseudo-UTP;1-(2,2,3,3,3-Pentafluoropropyl)pseudouridine TP;1-(2,2-Diethoxyethyl)pseudouridine TP;1-(2,4,6-Trimethylbenzyl)pseudouridine TP;1-(2,4,6-Trimethyl-benzyl)pseudo-UTP;1-(2,4,6-Trimethyl-phenyl)pseudo-UTP;1-(2-Amino-2-carboxyethyl)pseudo-UTP; 1-(2-Amino-ethyl)pseudo-UTP;1-(2-Hydroxyethyl)pseudouridine TP; 1-(2-Methoxyethyl)pseudouridine TP;1-(3,4-Bis-trifluoromethoxybenzyl)pseudouridine TP;1-(3,4-Dimethoxybenzyl)pseudouridine TP;1-(3-Amino-3-carboxypropyl)pseudo-UTP; 1-(3-Amino-propyl)pseudo-UTP;1-(3-Cyclopropyl-prop-2-ynyl)pseudouridine TP;1-(4-Amino-4-carboxybutyl)pseudo-UTP; 1-(4-Amino-benzyl)pseudo-UTP;1-(4-Amino-butyl)pseudo-UTP; 1-(4-Amino-phenyl)pseudo-UTP;1-(4-Azidobenzyl)pseudouridine TP; 1-(4-Bromobenzyl)pseudouridine TP;1-(4-Chlorobenzyl)pseudouridine TP; 1-(4-Fluorobenzyl)pseudouridine TP;1-(4-Iodobenzyl)pseudouridine TP;1-(4-Methanesulfonylbenzyl)pseudouridine TP;1-(4-Methoxybenzyl)pseudouridine TP; 1-(4-Methoxy-benzyl)pseudo-UTP;1-(4-Methoxy-phenyl)pseudo-UTP; 1-(4-Methylbenzyl)pseudouridine TP;1-(4-Methyl-benzyl)pseudo-UTP; 1-(4-Nitrobenzyl)pseudouridine TP;1-(4-Nitro-benzyl)pseudo-UTP; 1(4-Nitro-phenyl)pseudo-UTP;1-(4-Thiomethoxybenzyl)pseudouridine TP;1-(4-Trifluoromethoxybenzyl)pseudouridine TP;1-(4-Trifluoromethylbenzyl)pseudouridine TP;1-(5-Amino-pentyl)pseudo-UTP; 1-(6-Amino-hexyl)pseudo-UTP;1,6-Dimethyl-pseudo-UTP;1-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouridineTP; 1-{3-[2-(2-Aminoethoxy)-ethoxy]-propionyl}pseudouridine TP;1-Acetylpseudouridine TP; 1-Alkyl-6-(1-propynyl)-pseudo-UTP;1-Alkyl-6-(2-propynyl)-pseudo-UTP; 1-Alkyl-6-allyl-pseudo-UTP;1-Alkyl-6-ethynyl-pseudo-UTP; 1-Alkyl-6-homoallyl-pseudo-UTP;1-Alkyl-6-vinyl-pseudo-UTP; 1-Allylpseudouridine TP;1-Aminomethyl-pseudo-UTP; 1-Benzoylpseudouridine TP;1-Benzyloxymethylpseudouridine TP; 1-Benzyl-pseudo-UTP;1-Biotinyl-PEG2-pseudouridine TP; 1-Biotinylpseudouridine TP;1-Butyl-pseudo-UTP; 1-Cyanomethylpseudouridine TP;1-Cyclobutylmethyl-pseudo-UTP; 1-Cyclobutyl-pseudo-UTP;1-Cycloheptylmethyl-pseudo-UTP; 1-Cycloheptyl-pseudo-UTP;1-Cyclohexylmethyl-pseudo-UTP; 1-Cyclohexyl-pseudo-UTP;1-Cyclooctylmethyl-pseudo-UTP; 1-Cyclooctyl-pseudo-UTP;1-Cyclopentylmethyl-pseudo-UTP; 1-Cyclopentyl-pseudo-UTP;1-Cyclopropylmethyl-pseudo-UTP; 1-Cyclopropyl-pseudo-UTP;1-Ethyl-pseudo-UTP; 1-Hexyl-pseudo-UTP; 1-Homoallylpseudouridine TP;1-Hydroxymethylpseudouridine TP; 1-iso-propyl-pseudo-UTP;1-Me-2-thio-pseudo-UTP; 1-Me-4-thio-pseudo-UTP;1-Me-alpha-thio-pseudo-UTP; 1-Methanesulfonylmethylpseudouridine TP;1-Methoxymethylpseudouridine TP;1-Methyl-6-(2,2,2-Trifluoroethyl)pseudo-UTP;1-Methyl-6-(4-morpholino)-pseudo-UTP;1-Methyl-6-(4-thiomorpholino)-pseudo-UTP; 1-Methyl-6-(substitutedphenyl)pseudo-UTP; 1-Methyl-6-amino-pseudo-UTP;1-Methyl-6-azido-pseudo-UTP; 1-Methyl-6-bromo-pseudo-UTP;1-Methyl-6-butyl-pseudo-UTP; 1-Methyl-6-chloro-pseudo-UTP;1-Methyl-6-cyano-pseudo-UTP; 1-Methyl-6-dimethylamino-pseudo-UTP;1-Methyl-6-ethoxy-pseudo-UTP; 1-Methyl-6-ethylcarboxylate-pseudo-UTP;1-Methyl-6-ethyl-pseudo-UTP; 1-Methyl-6-fluoro-pseudo-UTP;1-Methyl-6-formyl-pseudo-UTP; 1-Methyl-6-hydroxyamino-pseudo-UTP;1-Methyl-6-hydroxy-pseudo-UTP; 1-Methyl-6-iodo-pseudo-UTP;1-Methyl-6-iso-propyl-pseudo-UTP; 1-Methyl-6-methoxy-pseudo-UTP;1-Methyl-6-methylamino-pseudo-UTP; 1-Methyl-6-phenyl-pseudo-UTP;1-Methyl-6-propyl-pseudo-UTP; 1-Methyl-6-tert-butyl-pseudo-UTP;1-Methyl-6-trifluoromethoxy-pseudo-UTP;1-Methyl-6-trifluoromethyl-pseudo-UTP; 1-MorpholinomethylpseudouridineTP; 1-Pentyl-pseudo-UTP; 1-Phenyl-pseudo-UTP; 1-PivaloylpseudouridineTP; 1-Propargylpseudouridine TP; 1-Propyl-pseudo-UTP;1-propynyl-pseudouridine; 1-p-tolyl-pseudo-UTP; 1-tert-Butyl-pseudo-UTP;1-Thiomethoxymethylpseudouridine TP; 1-Thiomorpholinomethylpseudouridine TP; 1-Trifluoroacetylpseudouridine TP;1-Trifluoromethyl-pseudo-UTP; 1-Vinylpseudouridine TP;2,2′-anhydro-uridine TP; 2′-bromo-deoxyuridine TP;2′-F-5-Methyl-2′-deoxy-UTP; 2′-OMe-5-Me-UTP; 2′-OMe-pseudo-UTP;2′-a-Ethynyluridine TP; 2′-a-Trifluoromethyluridine TP;2′-b-Ethynyluridine TP; 2′-b-Trifluoromethyluridine TP;2′-Deoxy-2′,2′-difluorouridine TP; 2′-Deoxy-2′-a-mercaptouridine TP;2′-Deoxy-2′-a-thiomethoxyuridine TP; 2′-Deoxy-2′-b-aminouridine TP;2′-Deoxy-2′-b-azidouridine TP; 2′-Deoxy-2′-b-bromouridine TP;2′-Deoxy-2′-b-chlorouridine TP; 2′-Deoxy-2′-b-fluorouridine TP;2′-Deoxy-2′-b-iodouridine TP; 2′-Deoxy-2′-b-mercaptouridine TP;2′-Deoxy-2′-b-thiomethoxyuridine TP; 2-methoxy-4-thio-uridine;2-methoxyuridine; 2′-O-Methyl-5-(1-propynyl)uridine TP;3-Alkyl-pseudo-UTP; 4′-Azidouridine TP; 4′-Carbocyclic uridine TP;4′-Ethynyluridine TP; 5-(1-Propynyl)ara-uridine TP; 5-(2-Furanyl)uridineTP; 5-Cyanouridine TP; 5-Dimethylaminouridine TP; 5′-Homo-uridine TP;5-iodo-2′-fluoro-deoxyuridine TP; 5-Phenylethynyluridine TP;5-Trideuteromethyl-6-deuterouridine TP; 5-Trifluoromethyl-Uridine TP;5-Vinylarauridine TP; 6-(2,2,2-Trifluoroethyl)-pseudo-UTP;6-(4-Morpholino)-pseudo-UTP; 6-(4-Thiomorpholino)-pseudo-UTP;6-(Substituted-Phenyl)-pseudo-UTP; 6-Amino-pseudo-UTP;6-Azido-pseudo-UTP; 6-Bromo-pseudo-UTP; 6-Butyl-pseudo-UTP;6-Chloro-pseudo-UTP; 6-Cyano-pseudo-UTP; 6-Dimethylamino-pseudo-UTP;6-Ethoxy-pseudo-UTP; 6-Ethylcarboxylate-pseudo-UTP; 6-Ethyl-pseudo-UTP;6-Fluoro-pseudo-UTP; 6-Formyl-pseudo-UTP; 6-Hydroxyamino-pseudo-UTP;6-Hydroxy-pseudo-UTP; 6-Iodo-pseudo-UTP; 6-iso-Propyl-pseudo-UTP;6-Methoxy-pseudo-UTP; 6-Methylamino-pseudo-UTP; 6-Methyl-pseudo-UTP;6-Phenyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Propyl-pseudo-UTP;6-tert-Butyl-pseudo-UTP; 6-Trifluoromethoxy-pseudo-UTP;6-Trifluoromethyl-pseudo-UTP; Alpha-thio-pseudo-UTP; Pseudouridine1-(4-methylbenzenesulfonic acid) TP; Pseudouridine 1-(4-methylbenzoicacid) TP; Pseudouridine TP 1-[3-(2-ethoxy)]propionic acid; PseudouridineTP 1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid;Pseudouridine TP1-[3-{2-(2-[2-{2(2-ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}]propionicacid; Pseudouridine TP 1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionicacid; Pseudouridine TP 1-[3-{2-(2-ethoxy)-ethoxy}] propionic acid;Pseudouridine TP 1-methylphosphonic acid; Pseudouridine TP1-methylphosphonic acid diethyl ester; Pseudo-UTP-N1-3-propionic acid;Pseudo-UTP-N1-4-butanoic acid; Pseudo-UTP-N1-5-pentanoic acid;Pseudo-UTP-N1-6-hexanoic acid; Pseudo-UTP-N1-7-heptanoic acid;Pseudo-UTP-N1-methyl-p-benzoic acid; Pseudo-UTP-N1-p-benzoic acid;Wybutosine; Hydroxywybutosine; Isowyosine; Peroxywybutosine;undermodified hydroxywybutosine; 4-demethylwyosine; 2,6-(diamino)purine;1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl:1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;1,3,5-(triaza)-2,6-(dioxa)-naphthalene; 2 (amino)purine;2,4,5-(trimethyl)phenyl; 2′ methyl, 2′amino, 2′azido, 2′fluro-cytidine;2′ methyl, 2′amino, 2′azido, 2′fluro-adenine; 2′methyl, 2′amino,2′azido, 2′fluro-uridine; 2′-amino-2′-deoxyribose;2-amino-6-Chloro-purine; 2-aza-inosinyl; 2′-azido-2′-deoxyribose;2′fluoro-2′-deoxyribose; 2′-fluoro-modified bases; 2′-O-methyl-ribose;2-oxo-7-aminopyridopyrimidin-3-yl; 2-oxo-pyridopyrimidine-3-yl;2-pyridinone; 3 nitropyrrole; 3-(methyl)-7-(propynyl)isocarbostyrilyl;3-(methyl)isocarbostyrilyl; 4-(fluoro)-6-(methyl)benzimidazole;4-(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; 5nitroindole; 5 substituted pyrimidines; 5-(methyl)isocarbostyrilyl;5-nitroindole; 6-(aza)pyrimidine; 6-(azo)thymine;6-(methyl)-7-(aza)indolyl; 6-chloro-purine;6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(aza)indolyl;7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazinl-yl;7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(propynyl)isocarbostyrilyl; 7-(propynyl)isocarbostyrilyl,propynyl-7-(aza)indolyl; 7-deaza-inosinyl; 7-substituted1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-substituted1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 9-(methyl)-imidizopyridinyl;Aminoindolyl; Anthracenyl;bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;bis-ortho-sub stituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;Difluorotolyl; Hypoxanthine; Imidizopyridinyl; Inosinyl;Isocarbostyrilyl; Isoguanisine; N2-substituted purines;N6-methyl-2-amino-purine; N6-substituted purines; N-alkylatedderivative; Napthalenyl; Nitrobenzimidazolyl; Nitroimidazolyl;Nitroindazolyl; Nitropyrazolyl; Nubularine; 06-substituted purines;O-alkylated derivative;ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;ortho-sub stituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Oxoformycin TP;para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Pentacenyl;Phenanthracenyl; Phenyl; propynyl-7-(aza)indolyl; Pyrenyl;pyridopyrimidin-3-yl; pyridopyrimidin-3-yl,2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-on-3-yl;Pyrrolopyrimidinyl; Pyrrolopyrizinyl; Stilbenzyl; substituted1,2,4-triazoles; Tetracenyl; Tubercidine; Xanthine; Xanthosine-5′-TP;2-thio-zebularine; 5-aza-2-thio-zebularine; 7-deaza-2-amino-purine;pyridin-4-one ribonucleoside; 2-Amino-riboside-TP; Formycin A TP;Formycin B TP; Pyrrolosine TP; 2′-OH-ara-adenosine TP;2′-OH-ara-cytidine TP; 2′-OH-ara-uridine TP; 2′-OH-ara-guanosine TP;5-(2-carbomethoxyvinyl)uridine TP; andN6-(19-Amino-pentaoxanonadecyl)adenosine TP.

In some embodiments, the polynucleotide (e.g., RNA polynucleotide, suchas mRNA polynucleotide) includes a combination of at least two (e.g., 2,3, 4 or more) of the aforementioned modified nucleobases.

In some embodiments, the mRNA comprises at least one chemically modifiednucleoside. In some embodiments, the at least one chemically modifiednucleoside is selected from the group consisting of pseudouridine (ψ),2-thiouridine (s2U), 4′-thiouridine, 5-methylcytosine,2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine,2-thio-5-aza-uridine, 2-thio-dihydropseudouridine,2-thio-dihydrouridine, 2-thio-pseudouridine,4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine,4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine,dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, 2′-O-methyluridine, 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ),5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), α-thio-guanosine,α-thio-adenosine, 5-cyano uridine, 4′-thio uridine 7-deaza-adenine,1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine(m6A), and 2,6-Diaminopurine, (I), 1-methyl-inosine (m1I), wyosine(imG), methylwyosine (mimG), 7-deaza-guanosine,7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine(preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G),8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 2,8-dimethyladenosine,2-geranylthiouridine, 2-lysidine, 2-selenouridine,3-(3-amino-3-carboxypropyl)-5,6-dihydrouridine,3-(3-amino-3-carboxypropyl)pseudouridine, 3-methylpseudouridine,5-(carboxyhydroxymethyl)-2′-O-methyluridine methyl ester,5-aminomethyl-2-geranylthiouridine, 5-aminomethyl-2-selenouridine,5-aminomethyluridine, 5-carbamoylhydroxymethyluridine,5-carbamoylmethyl-2-thiouridine, 5-carboxymethyl-2-thiouridine,5-carboxymethylaminomethyl-2-geranylthiouridine,5-carboxymethylaminomethyl-2-selenouridine, 5-cyanomethyluridine,5-hydroxycytidine, 5-methylaminomethyl-2-geranylthiouridine,7-aminocarboxypropyl-demethylwyosine, 7-aminocarboxypropylwyosine,7-aminocarboxypropylwyosine methyl ester, 8-methyladenosine,N4,N4-dimethylcytidine, N6-formyladenosine, N6-hydroxymethyladenosine,agmatidine, cyclic N6-threonylcarbamoyladenosine, glutamyl-queuosine,methylated undermodified hydroxywybutosine,N4,N4,2′-O-trimethylcytidine, geranylated5-methylaminomethyl-2-thiouridine, geranylated5-carboxymethylaminomethyl-2-thiouridine, Qbase, preQ0base, preQ1base,and two or more combinations thereof. In some embodiments, the at leastone chemically modified nucleoside is selected from the group consistingof pseudouridine, 1-methyl-pseudouridine, 1-ethyl-pseudouridine,5-methylcytosine, 5-methoxyuridine, and a combination thereof. In someembodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNApolynucleotide) includes a combination of at least two (e.g., 2, 3, 4 ormore) of the aforementioned modified nucleobases.

Base Modifications

In certain embodiments, the chemical modification is at nucleobases inthe polynucleotides (e.g., RNA polynucleotide, such as mRNApolynucleotide). In some embodiments, modified nucleobases in thepolynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide)are selected from the group consisting of 1-methyl-pseudouridine (m1ψ),1-ethyl-pseudouridine (e1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine(m5C), pseudouridine (ψ), α-thio-guanosine and α-thio-adenosine. In someembodiments, the polynucleotide includes a combination of at least two(e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.

In some embodiments, the polynucleotide (e.g., RNA polynucleotide, suchas mRNA polynucleotide) comprises pseudouridine (ψ) and5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g.,RNA polynucleotide, such as mRNA polynucleotide) comprises1-methyl-pseudouridine (m1ψ). In some embodiments, the polynucleotide(e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises1-ethyl-pseudouridine (e1ψ). In some embodiments, the polynucleotide(e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises1-methyl-pseudouridine (m1ψ) and 5-methyl-cytidine (m5C). In someembodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNApolynucleotide) comprises 1-ethyl-pseudouridine (e1ψ) and5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g.,RNA polynucleotide, such as mRNA polynucleotide) comprises 2-thiouridine(s2U). In some embodiments, the polynucleotide (e.g., RNApolynucleotide, such as mRNA polynucleotide) comprises 2-thiouridine and5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g.,RNA polynucleotide, such as mRNA polynucleotide) comprisesmethoxy-uridine (mo5U). In some embodiments, the polynucleotide (e.g.,RNA polynucleotide, such as mRNA polynucleotide) comprises5-methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C). In someembodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNApolynucleotide) comprises 2′-O-methyl uridine. In some embodiments, thepolynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide)comprises 2′-O-methyl uridine and 5-methyl-cytidine (m5C). In someembodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNApolynucleotide) comprises N6-methyl-adenosine (m6A). In someembodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNApolynucleotide) comprises N6-methyl-adenosine (m6A) and5-methyl-cytidine (m5C).

In some embodiments, the polynucleotide (e.g., RNA polynucleotide, suchas mRNA polynucleotide) is uniformly modified (e.g., fully modified,modified throughout the entire sequence) for a particular modification.For example, a polynucleotide can be uniformly modified with5-methyl-cytidine (m5C), meaning that all cytosine residues in the mRNAsequence are replaced with 5-methyl-cytidine (m5C). Similarly, apolynucleotide can be uniformly modified for any type of nucleosideresidue present in the sequence by replacement with a modified residuesuch as any of those set forth above.

In some embodiments, the chemically modified nucleosides in the openreading frame are selected from the group consisting of uridine,adenine, cytosine, guanine, and any combination thereof.

In some embodiments, the modified nucleobase is a modified cytosine.Examples of nucleobases and nucleosides having a modified cytosineinclude N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C),5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine(hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C),2-thio-5-methyl-cytidine.

In some embodiments, a modified nucleobase is a modified uridine.Example nucleobases and nucleosides having a modified uridine include5-cyano uridine or 4′-thio uridine.

In some embodiments, a modified nucleobase is a modified adenine.Example nucleobases and nucleosides having a modified adenine include7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A),N6-methyl-adenine (m6A), and 2,6-Diaminopurine.

In some embodiments, a modified nucleobase is a modified guanine.Example nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine(mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0),7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G),1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine.

In some embodiments, the nucleobase modified nucleotides in thepolynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide)are 5-methoxyuridine.

In some embodiments, the polynucleotide (e.g., RNA polynucleotide, suchas mRNA polynucleotide) includes a combination of at least two (e.g., 2,3, 4 or more) of modified nucleobases.

In some embodiments, at least 95% of a type of nucleobases (e.g.,uracil) in a polynucleotide of the disclosure (e.g., an mRNApolynucleotide encoding MCM) are modified nucleobases. In someembodiments, at least 95% of uracil in a polynucleotide of the presentdisclosure (e.g., an mRNA polynucleotide encoding MCM) is5-methoxyuracil.

In some embodiments, the polynucleotide (e.g., RNA polynucleotide, suchas mRNA polynucleotide) comprises 5-methoxyuridine (5mo5U) and5-methyl-cytidine (m5C).

In some embodiments, the polynucleotide (e.g., RNA polynucleotide, suchas mRNA polynucleotide) is uniformly modified (e.g., fully modified,modified throughout the entire sequence) for a particular modification.For example, a polynucleotide can be uniformly modified with5-methoxyuridine, meaning that substantially all uridine residues in themRNA sequence are replaced with 5-methoxyuridine. Similarly, apolynucleotide can be uniformly modified for any type of nucleosideresidue present in the sequence by replacement with a modified residuesuch as any of those set forth above.

In some embodiments, the modified nucleobase is a modified cytosine.

In some embodiments, a modified nucleobase is a modified uracil. Examplenucleobases and nucleosides having a modified uracil include5-methoxyuracil.

In some embodiments, a modified nucleobase is a modified adenine.

In some embodiments, a modified nucleobase is a modified guanine.

In some embodiments, the nucleobases, sugar, backbone, or anycombination thereof in the open reading frame encoding an MCMpolypeptide are chemically modified by at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 99%, or 100%.

In some embodiments, the uridine nucleosides in the open reading frameencoding an MCM polypeptide are chemically modified by at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 99%, or100%.

In some embodiments, the adenosine nucleosides in the open reading frameencoding an MCM polypeptide are chemically modified by at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 99%, or100%.

In some embodiments, the cytidine nucleosides in the open reading frameencoding an MCM polypeptide are chemically modified by at least at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, at least99%, or 100%.

In some embodiments, the guanosine nucleosides in the open reading frameencoding an MCM polypeptide are chemically modified by at least at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, at least99%, or 100%.

In some embodiments, the polynucleotides can include any useful linkerbetween the nucleosides. Such linkers, including backbone modifications,that are useful in the composition of the present disclosure include,but are not limited to the following: 3′-alkylene phosphonates, 3′-aminophosphoramidate, alkene containing backbones,aminoalkylphosphoramidates, aminoalkylphosphotriesters,boranophosphates, —CH₂—O—N(CH₃)—CH₂—, ˜CH₂—N(CH₃)—N(CH₃)—CH₂—,˜CH₂—NH—CH₂—, chiral phosphonates, chiral phosphorothioates, formacetyland thioformacetyl backbones, methylene (methylimino), methyleneformacetyl and thioformacetyl backbones, methyleneimino andmethylenehydrazino backbones, morpholino linkages, —N(CH₃)—CH₂˜CH₂—,oligonucleosides with heteroatom internucleoside linkage, phosphinates,phosphoramidates, phosphorodithioates, phosphorothioate internucleosidelinkages, phosphorothioates, phosphotriesters, PNA, siloxane backbones,sulfamate backbones, sulfide sulfoxide and sulfone backbones, sulfonateand sulfonamide backbones, thionoalkylphosphonates,thionoalkylphosphotriesters, and thionophosphoramidates.

Modifications on the Sugar

The modified nucleosides and nucleotides (e.g., building blockmolecules), which can be incorporated into a polynucleotide (e.g., RNAor mRNA, as described herein), can be modified on the sugar of theribonucleic acid. For example, the 2′ hydroxyl group (OH) can bemodified or replaced with a number of different substituents. Exemplarysubstitutions at the 2′-position include, but are not limited to, H,halo, optionally substituted C₁₋₆ alkyl; optionally substituted C₁₋₆alkoxy; optionally substituted C₆₋₁₀ aryloxy; optionally substitutedC₃₋₈ cycloalkyl; optionally substituted C₃₋₈ cycloalkoxy; optionallysubstituted C₆₋₁₀ aryloxy; optionally substituted C₆₋₁₀ aryl-C₁₋₆alkoxy, optionally substituted C₁₋₁₂ (heterocyclyl)oxy; a sugar (e.g.,ribose, pentose, or any described herein); a polyethyleneglycol (PEG),—O(CH₂CH₂O)_(n)CH₂CH₂OR, where R is H or optionally substituted alkyl,and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16,from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to20); “locked” nucleic acids (LNA) in which the 2′-hydroxyl is connectedby a C₁₋₆ alkylene or C₁₋₆ heteroalkylene bridge to the 4′-carbon of thesame ribose sugar, where exemplary bridges included methylene,propylene, ether, or amino bridges; aminoalkyl, as defined herein;aminoalkoxy, as defined herein; amino as defined herein; and amino acid,as defined herein

Generally, RNA includes the sugar group ribose, which is a 5-memberedring having an oxygen. Exemplary, non-limiting modified nucleotidesinclude replacement of the oxygen in ribose (e.g., with S, Se, oralkylene, such as methylene or ethylene); addition of a double bond(e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ringcontraction of ribose (e.g., to form a 4-membered ring of cyclobutane oroxetane); ring expansion of ribose (e.g., to form a 6- or 7-memberedring having an additional carbon or heteroatom, such as foranhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, andmorpholino that also has a phosphoramidate backbone); multicyclic forms(e.g., tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA)(e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attachedto phosphodiester bonds), threose nucleic acid (TNA, where ribose isreplace with α-L-threofuranosyl-(3′→2′)), and peptide nucleic acid (PNA,where 2-amino-ethyl-glycine linkages replace the ribose andphosphodiester backbone). The sugar group can also contain one or morecarbons that possess the opposite stereochemical configuration than thatof the corresponding carbon in ribose. Thus, a polynucleotide moleculecan include nucleotides containing, e.g., arabinose, as the sugar. Suchsugar modifications are taught International Patent Publication No.WO2013052523 and International Patent Application No. PCT/US2013/75177,the contents of each of which are incorporated herein by reference inits entirety.

Combinations of Modifications

The polynucleotides of the disclosure can include a combination ofmodifications to the sugar, the nucleobase, and/or the internucleosidelinkage. These combinations can include any one or more modificationsdescribed herein.

Examples of modified nucleotides and modified nucleotide combinationsare provided below in Table 4. These combinations of modifiednucleotides can be used to form the polynucleotides of the disclosure.Unless otherwise noted, the modified nucleotides can be completelysubstituted for the natural nucleotides of the polynucleotides of thedisclosure. As a non-limiting example, the natural nucleotide uridinecan be substituted with a modified nucleoside described herein. Inanother non-limiting example, the natural nucleotide uridine can bepartially substituted (e.g., about 0.1%, 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or99.9%) with at least one of the modified nucleoside disclosed herein.Any combination of base/sugar or linker can be incorporated into thepolynucleotides of the disclosure and such modifications are taught inInternational Patent Publication No. WO2013052523 and InternationalPatent Application No. PCT/US2013/75177, the contents of each of whichare incorporated herein by reference in its entirety.

TABLE 4 Combinations Uracil Cytosine Adenine Guanine 5-methoxy-UTP CTPATP GTP 5-Methoxy-UTP N4Ac-CTP ATP GTP 5-Methoxy-UTP 5-Methyl-CTP ATPGTP 5-Methoxy-UTP 5-Trifluoromethyl-CTP ATP GTP 5-Methoxy-UTP5-Hydroxymethyl-CTP ATP GTP 5-Methoxy-UTP 5-Bromo-CTP ATP GTP5-Methoxy-UTP N4Ac-CTP ATP GTP 5-Methoxy-UTP CTP ATP GTP 5-Methoxy-UTP5-Methyl-CTP ATP GTP 5-Methoxy-UTP 5-Trifluoromethyl-CTP ATP GTP5-Methoxy-UTP 5-Hydroxymethyl-CTP ATP GTP 5-Methoxy-UTP 5-Bromo-CTP ATPGTP 5-Methoxy-UTP N4—Ac-CTP ATP GTP 5-Methoxy-UTP 5-Iodo-CTP ATP GTP5-Methoxy-UTP 5-Bromo-CTP ATP GTP 5-Methoxy-UTP CTP ATP GTP5-Methoxy-UTP 5-Methyl-CTP ATP GTP 75% 5-Methoxy-UTP + 25% UTP5-Methyl-CTP ATP GTP 50% 5-Methoxy-UTP + 50% UTP 5-Methyl-CTP ATP GTP25% 5-Methoxy-UTP + 75% UTP 5-Methyl-CTP ATP GTP 5-Methoxy-UTP 75%5-Methyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTP 50% 5-Methyl-CTP + 50% CTPATP GTP 5-Methoxy-UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 50%5-Methoxy-UTP + 50% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 50%5-Methoxy-UTP + 50% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 50%5-Methoxy-UTP + 50% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP CTP ATP GTP 50% 5-Methoxy-UTP + 50% UTP CTP ATPGTP 25% 5-Methoxy-UTP + 75% UTP CTP ATP GTP 5-Methoxy-UTP CTP ATP GTP5-Methoxy-UTP CTP ATP GTP 5-Methoxy-UTP CTP ATP GTP 5-Methoxy-UTP5-Methyl-CTP ATP GTP 5-Methoxy-UTP 5-Methyl-CTP ATP GTP 5-Methoxy-UTP5-Methyl-CTP ATP GTP 5-Methoxy-UTP CTP Alpha-thio-ATP GTP 5-Methoxy-UTP5-Methyl-CTP Alpha-thio-ATP GTP 5-Methoxy-UTP CTP ATP Alpha-thio-GTP5-Methoxy-UTP 5-Methyl-CTP ATP Alpha-thio-GTP 5-Methoxy-UTP CTPN6—Me-ATP GTP 5-Methoxy-UTP 5-Methyl-CTP N6—Me-ATP GTP 5-Methoxy-UTP CTPATP GTP 5-Methoxy-UTP 5-Methyl-CTP ATP GTP 75% 5-Methoxy-UTP + 25% UTP5-Methyl-CTP ATP GTP 50% 5-Methoxy-UTP + 50% UTP 5-Methyl-CTP ATP GTP25% 5-Methoxy-UTP + 75% UTP 5-Methyl-CTP ATP GTP 5-Methoxy-UTP 75%5-Methyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTP 50% 5-Methyl-CTP + 50% CTPATP GTP 5-Methoxy-UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 50%5-Methoxy-UTP + 50% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 50%5-Methoxy-UTP + 50% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 50%5-Methoxy-UTP + 50% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP CTP ATP GTP 50% 5-Methoxy-UTP + 50% UTP CTP ATPGTP 25% 5-Methoxy-UTP + 75% UTP CTP ATP GTP 5-Methoxy-UTP 5-Ethyl-CTPATP GTP 5-Methoxy-UTP 5-Methoxy-CTP ATP GTP 5-Methoxy-UTP 5-Ethynyl-CTPATP GTP 5-Methoxy-UTP CTP ATP GTP 5-Methoxy-UTP 5-Methyl-CTP ATP GTP5-Methoxy-UTP CTP ATP GTP 5-Methoxy-UTP 5-Methyl-CTP ATP GTP 75%5-Methoxy-UTP + 25% 1- 5-Methyl-CTP ATP GTP Methyl-pseudo-UTP 50%5-Methoxy-UTP + 50% 1- 5-Methyl-CTP ATP GTP Methyl-pseudo-UTP 25%5-Methoxy-UTP + 75% 1- 5-Methyl-CTP ATP GTP Methyl-pseudo-UTP5-Methoxy-UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTP 50%5-Methyl-CTP + 50% CTP ATP GTP 5-Methoxy-UTP 25% 5-Methyl-CTP + 75% CTPATP GTP 75% 5-Methoxy-UTP + 25% 1- 75% 5-Methyl-CTP + 25% CTP ATP GTPMethyl-pseudo-UTP 75% 5-Methoxy-UTP + 25% 1- 50% 5-Methyl-CTP + 50% CTPATP GTP Methyl-pseudo-UTP 75% 5-Methoxy-UTP + 25% 1- 25% 5-Methyl-CTP +75% CTP ATP GTP Methyl-pseudo-UTP 50% 5-Methoxy-UTP + 50% 1- 75%5-Methyl-CTP + 25% CTP ATP GTP Methyl-pseudo-UTP 50% 5-Methoxy-UTP + 50%1- 50% 5-Methyl-CTP + 50% CTP ATP GTP Methyl-pseudo-UTP 50%5-Methoxy-UTP + 50% 1- 25% 5-Methyl-CTP + 75% CTP ATP GTPMethyl-pseudo-UTP 25% 5-Methoxy-UTP + 75% 1- 75% 5-Methyl-CTP + 25% CTPATP GTP Methyl-pseudo-UTP 25% 5-Methoxy-UTP + 75% 1- 50% 5-Methyl-CTP +50% CTP ATP GTP Methyl-pseudo-UTP 25% 5-Methoxy-UTP + 75% 1- 25%5-Methyl-CTP + 75% CTP ATP GTP Methyl-pseudo-UTP 75% 5-Methoxy-UTP + 25%1- CTP ATP GTP Methyl-pseudo-UTP 50% 5-Methoxy-UTP + 50% 1- CTP ATP GTPMethyl-pseudo-UTP 25% 5-Methoxy-UTP + 75% 1- CTP ATP GTPMethyl-pseudo-UTP 5-methoxy-UTP CTP ATP GTP 5-methoxy-UTP CTP ATP GTP5-methoxy-UTP 5-Methyl-CTP ATP GTP 5-Methoxy-UTP CTP ATP GTP5-Methoxy-UTP 5-Methyl-CTP ATP GTP 75% 5-Methoxy-UTP + 25% UTP5-Methyl-CTP ATP GTP 50% 5-Methoxy-UTP + 50% UTP 5-Methyl-CTP ATP GTP25% 5-Methoxy-UTP + 75% UTP 5-Methyl-CTP ATP GTP 5-Methoxy-UTP 75%5-Methyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTP 50% 5-Methyl-CTP + 50% CTPATP GTP 5-Methoxy-UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 50%5-Methoxy-UTP + 50% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 50%5-Methoxy-UTP + 50% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 50%5-Methoxy-UTP + 50% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP CTP ATP GTP 50% 5-Methoxy-UTP + 50% UTP CTP ATPGTP 25% 5-Methoxy-UTP + 75% UTP CTP ATP GTP 5-Methoxy-UTP CTP ATP GTP5-Methoxy-UTP 5-Methyl-CTP ATP GTP 75% 5-Methoxy-UTP + 25% UTP5-Methyl-CTP ATP GTP 50% 5-Methoxy-UTP + 50% UTP 5-Methyl-CTP ATP GTP25% 5-Methoxy-UTP + 75% UTP 5-Methyl-CTP ATP GTP 5-Methoxy-UTP 75%5-Methyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTP 50% 5-Methyl-CTP + 50% CTPATP GTP 5-Methoxy-UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 50%5-Methoxy-UTP + 50% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 50%5-Methoxy-UTP + 50% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 50%5-Methoxy-UTP + 50% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP CTP ATP GTP 50% 5-Methoxy-UTP + 50% UTP CTP ATPGTP 25% 5-Methoxy-UTP + 75% UTP CTP ATP GTP 5-Methoxy-UTP CTP ATP GTP25% 5-Methoxy-UTP + 75% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTPCTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 75% 5-Methyl-CTP + 25% CTP ATPGTP 25% 5-Methoxy-UTP + 75% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTPCTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 75% 5-Methyl-CTP + 25% CTP ATPGTP 25% 5-Methoxy-UTP + 75% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTPCTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 75% 5-Methyl-CTP + 25% CTP ATPGTP 25% 5-Methoxy-UTP + 75% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTPCTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 75% 5-Methyl-CTP + 25% CTP ATPGTP 25% 5-Methoxy-UTP + 75% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTPCTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 75% 5-Methyl-CTP + 25% CTP ATPGTP 25% 5-Methoxy-UTP + 75% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTPCTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 75% 5-Methyl-CTP + 25% CTP ATPGTP 25% 5-Methoxy-UTP + 75% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTP5-Fluoro-CTP ATP GTP 5-Methoxy-UTP 5-Phenyl-CTP ATP GTP 5-Methoxy-UTPN4—Bz-CTP ATP GTP 5-Methoxy-UTP CTP N6-Isopentenyl- GTP ATP5-Methoxy-UTP N4—Ac-CTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 25%N4—Ac-CTP + 75% CTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 75% N4—Ac-CTP +25% CTP ATP GTP 75% 5-Methoxy-UTP + 25% UTP 25% N4—Ac-CTP + 75% CTP ATPGTP 75% 5-Methoxy-UTP + 25% UTP 75% N4—Ac-CTP + 25% CTP ATP GTP5-Methoxy-UTP 5-Hydroxymethyl-CTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP25% 5-Hydroxymethyl-CTP + 75% ATP GTP CTP 25% 5-Methoxy-UTP + 75% UTP75% 5-Hydroxymethyl-CTP + 25% ATP GTP CTP 75% 5-Methoxy-UTP + 25% UTP25% 5-Hydroxymethyl-CTP + 75% ATP GTP CTP 75% 5-Methoxy-UTP + 25% UTP75% 5-Hydroxymethyl-CTP + 25% ATP GTP CTP 5-Methoxy-UTP N4-Methyl CTPATP GTP 25% 5-Methoxy-UTP + 75% UTP 25% N4-Methyl CTP + 75% CTP ATP GTP25% 5-Methoxy-UTP + 75% UTP 75% N4-Methyl CTP + 25% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 25% N4-Methyl CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% N4-Methyl CTP + 25% CTP ATP GTP5-Methoxy-UTP 5-Trifluoromethyl-CTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP25% 5-Trifluoromethyl-CTP + 75% ATP GTP CTP 25% 5-Methoxy-UTP + 75% UTP75% 5-Trifluoromethyl-CTP + 25% ATP GTP CTP 75% 5-Methoxy-UTP + 25% UTP25% 5-Trifluoromethyl-CTP + 75% ATP GTP CTP 75% 5-Methoxy-UTP + 25% UTP75% 5-Trifluoromethyl-CTP + 25% ATP GTP CTP 5-Methoxy-UTP 5-Bromo-CTPATP GTP 25% 5-Methoxy-UTP + 75% UTP 25% 5-Bromo-CTP + 75% CTP ATP GTP25% 5-Methoxy-UTP + 75% UTP 75% 5-Bromo-CTP + 25% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 25% 5-Bromo-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Bromo-CTP + 25% CTP ATP GTP 5-Methoxy-UTP5-Iodo-CTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 25% 5-Iodo-CTP + 75% CTPATP GTP 25% 5-Methoxy-UTP + 75% UTP 75% 5-Iodo-CTP + 25% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 25% 5-Iodo-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Iodo-CTP + 25% CTP ATP GTP 5-Methoxy-UTP5-Ethyl-CTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 25% 5-Ethyl-CTP + 75%CTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 75% 5-Ethyl-CTP + 25% CTP ATPGTP 75% 5-Methoxy-UTP + 25% UTP 25% 5-Ethyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Ethyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTP5-Methoxy-CTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 25% 5-Methoxy-CTP +75% CTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 75% 5-Methoxy-CTP + 25% CTPATP GTP 75% 5-Methoxy-UTP + 25% UTP 25% 5-Methoxy-CTP + 75% CTP ATP GTP75% 5-Methoxy-UTP + 25% UTP 75% 5-Methoxy-CTP + 25% CTP ATP GTP5-Methoxy-UTP 5-Ethynyl-CTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 25%5-Ethynyl-CTP + 75% CTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 75%5-Ethynyl-CTP + 25% CTP ATP GTP 75% 5-Methoxy-UTP + 25% UTP 25%5-Ethynyl-CTP + 75% CTP ATP GTP 75% 5-Methoxy-UTP + 25% UTP 75%5-Ethynyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTP 5-Pseudo-iso-CTP ATP GTP25% 5-Methoxy-UTP + 75% UTP 25% 5-Pseudo-iso-CTP + 75% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 75% 5-Pseudo-iso-CTP + 25% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 25% 5-Pseudo-iso-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Pseudo-iso-CTP + 25% CTP ATP GTP5-Methoxy-UTP 5-Formyl-CTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 25%5-Formyl-CTP + 75% CTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 75%5-Formyl-CTP + 25% CTP ATP GTP 75% 5-Methoxy-UTP + 25% UTP 25%5-Formyl-CTP + 75% CTP ATP GTP 75% 5-Methoxy-UTP + 25% UTP 75%5-Formyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTP 5-Aminoallyl-CTP ATP GTP25% 5-Methoxy-UTP + 75% UTP 25% 5-Aminoallyl-CTP + 75% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 75% 5-Aminoallyl-CTP + 25% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 25% 5-Aminoallyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Aminoallyl-CTP + 25% CTP ATP GTP

III. POLYNUCLEOTIDE ARCHITECTURE

Traditionally, the basic components of an mRNA molecule include at leasta coding region, a 5′UTR, a 3′UTR, a 5′ cap and a poly-A tail. Thepolynucleotides of the present disclosure can function as mRNA but aredistinguished from wild-type mRNA in their functional and/or structuraldesign features that serve, e.g., to overcome existing problems ofeffective polypeptide production using nucleic-acid based therapeutics.

In Vitro Transcribed Polynucleotides

The disclosure also includes an in vitro transcribed polynucleotidecomprising the polynucleotide described herein, i.e., a polynucleotidecomprising an ORF encoding an MCM polypeptide.

Polynucleotides which are made using only in vitro transcription (IVT)enzymatic synthesis methods are referred to as “IVT polynucleotides.”Methods of making IVT polynucleotides are known in the art and aredescribed, e.g., in International Publication Nos. WO2013151666,WO2013151667, WO2013151668, WO2013151663, WO2013151669, WO2013151670,WO2013151664, WO2013151665, WO2013151671, WO2013151672 and WO2013151736;the contents of each of which are herein incorporated by reference intheir entireties.

The shortest length of the first region of the primary construct of theIVT polynucleotide can be the length of a nucleic acid sequence that issufficient to encode for MCM, a fragment thereof, or variant thereof.The length of the first region of the primary construct of the IVTpolynucleotide encoding the polypeptide of interest can be greater thanabout 30 nucleotides in length (e.g., at least or greater than about2,154, 2,250, 2,500, and 3,000, 4,000, 4,100, 4,200, 4,300, 4,400,4,500, 4,600, 4,700, 4,800, 4,900, 5,000, 5,100, 5,200, 5,300, 5,400,5,500, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000,50,000, 60,000, 70,000, 80,000, 90,000 or up to and including 100,000nucleotides).

In some embodiments, the first and second flanking regions of the IVTpolynucleotide can range independently from 15-1,000 nucleotides inlength (e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120,140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 5,500nucleotides or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120,140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 5,500nucleotides).

In some embodiments, the tailing sequence of the IVT polynucleotide canrange from absent to 500 nucleotides in length (e.g., at least 60, 70,80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500nucleotides). Where the tailing region is a polyA tail, the length canbe determined in units of or as a function of polyA Binding Proteinbinding. In this embodiment, the polyA tail is long enough to bind atleast 4 monomers of PolyA Binding Protein. PolyA Binding Proteinmonomers bind to stretches of approximately 38 nucleotides. As such, ithas been observed that polyA tails of about 80 nucleotides and 160nucleotides are functional.

In some embodiments, the capping region of the IVT polynucleotide cancomprise a single cap or a series of nucleotides forming the cap. Inthis embodiment the capping region can be from 1 to 10, e.g., 2-9, 3-8,4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length. Insome embodiments, the cap is absent.

In some embodiments, the first and second operational regions of the IVTpolynucleotide can range from 3 to 40, e.g., 5-30, 10-20, 15, or atleast 4, or 30 or fewer nucleotides in length and can comprise, inaddition to a Start and/or Stop codon, one or more signal and/orrestriction sequences.

In some embodiments, the IVT polynucleotides can be structurallymodified or chemically modified. When the IVT polynucleotides arechemically and/or structurally modified, the polynucleotides can bereferred to as “modified IVT polynucleotides.”

In some embodiments, if the IVT polynucleotides are chemically modifiedthey can have a uniform chemical modification of all or any of the samenucleoside type or a population of modifications produced by meredownward titration of the same starting modification in all or any ofthe same nucleoside type, or a measured percent of a chemicalmodification of all any of the same nucleoside type but with randomincorporation, such as where all uridines are replaced by a uridineanalog, e.g., pseudouridine or 5-methoxyuridine. In another embodiment,the IVT polynucleotides can have a uniform chemical modification of two,three, or four of the same nucleoside type throughout the entirepolynucleotide (such as all uridines and all cytosines, etc. aremodified in the same way).

In some embodiments, the IVT polynucleotide can encode MCM and at leastone additional peptide or polypeptide of interest. In anotherembodiment, the IVT polynucleotide can encode MCM and two or morepeptides or polypeptides of interest. Non-limiting examples of peptidesor polypeptides of interest include an enzyme and its substrate, a labeland its binding molecule, a second messenger and its enzyme or thecomponents of multimeric proteins or complexes.

In some embodiments, the IVT polynucleotide encodes an MCM protein or afunctional fragment thereof. In some embodiments, the IVTpolynucleotides of the disclosure comprise any one of the human MCMnucleic acid sequences selected from SEQ ID NOs: 1 to 207, 732 to 765,and 772. In some embodiments, the IVT polynucleotide encodes a human MCMor functional fragment thereof comprising at least one amino acidmutation from the wild type sequence. In some embodiments, the IVTpolynucleotide encodes an MCM mutant comprising one or more of the pointmutations V69, T499, H532, A598, and V671. In some embodiments, theexpression of the encoded polypeptide is increased. In some embodiments,the IVT polynucleotide increases MCM expression levels in cells whenintroduced into those cells, e.g., by 20-50%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least50%.

Chimeric Polynucleotide Architecture

The disclosure also includes a chimeric polynucleotide comprising thepolynucleotide described herein, i.e., a polynucleotide comprising anORF encoding an MCM polypeptide.

Polynucleotides which have portions or regions which differ in sizeand/or chemical modification pattern, chemical modification position,chemical modification percent or chemical modification population andcombinations of the foregoing are known as “chimeric polynucleotides.” A“chimera” according to the present disclosure is an entity having two ormore incongruous or heterogeneous parts or regions. As used herein a“part” or “region” of a polynucleotide is defined as any portion of thepolynucleotide which is less than the entire length of thepolynucleotide. Chimeric polynucleotides which are modified mRNAmolecules are termed “chimeric modified mRNA” or “chimeric mRNA.”

Chimeric polynucleotides have portions or regions that differ in sizeand/or chemical modification pattern, chemical modification position,chemical modification percent or chemical modification population andcombinations of the foregoing.

Examples of parts or regions, where the chimeric polynucleotidefunctions as an mRNA and encodes a polypeptide of interest include, butare not limited to, untranslated regions (UTRs, such as the 5′ UTR or 3′UTR), coding regions, cap regions, polyA tail regions, start regions,stop regions, signal or target sequence regions, and combinationsthereof.

In some embodiments, the chimeric polynucleotides of the disclosure havea structure comprising Formula I.

5′[A_(n)]_(x)-L1-[B_(o)]_(y)-L2-[C_(p)]_(z)-L3 3′   Formula I

wherein:each of A and B independently comprise a region of linked nucleosides;either A or B or both A and B encode MCM as described elsewhere herein;C is an optional region of linked nucleosides;at least one of regions A, B, or C is positionally modified, whereinsaid positionally modified region comprises at least two chemicallymodified nucleosides of one or more of the same nucleoside type ofadenosine, thymidine, guanosine, cytidine, or uridine, and wherein atleast two of the chemical modifications of nucleosides of the same typeare different chemical modifications;n, o and p are independently an integer between 15-10,000, representingthe number of nucleosides in regions A, B, and C, respectively;x and y are independently 1-20;z is 0-5;L1 and L2 are independently optional linker moieties, said linkermoieties being either nucleic acid based or non-nucleic acid based; andL3 is an optional conjugate or an optional linker moiety, said linkermoiety being either nucleic acid based or non-nucleic acid based.

In some embodiments, at least one of the regions of linked nucleosidesof A can comprise a sequence of linked nucleosides that can function asa 5′ untranslated region (UTR). The sequence of linked nucleosides canbe a natural or synthetic 5′ UTR. As a non-limiting example, thechimeric polynucleotide can encode MCM and the sequence of linkednucleosides of A can encode the native 5′ UTR of the MCM protein or anon-heterologous 5′ UTR such as, but not limited to a synthetic UTR.

In another embodiment, at least one of the regions of linked nucleosidesof A can be a cap region. The cap region can be located 5′ to a regionof linked nucleosides of A functioning as a 5′UTR. The cap region cancomprise at least one cap such as, but not limited to, Cap0, Cap1, ARCA,inosine, N1-methyl-guanosine, 2′fluoro-guanosine, 7-deaza-guanosine,8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine,Cap2 and Cap4.

In some embodiments, the polynucleotide of the disclosure comprises aCap1 5′UTR. In some embodiments, a polynucleotide comprises the Cap15′UTR, wherein the polynucleotide encodes human MCM or functionalfragment thereof. In some embodiments, a polynucleotide comprising 5′UTRsequence, e.g., Cap1, for encoding an MCM protein as disclosed hereinincreases expression of MCM compared to polynucleotides encoding MCMcomprising a different 5′UTR (e.g., Cap0, ARCA, inosine,N1-methyl-guanosine, 2′fluoro-guanosine, 7-deaza-guanosine,8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine,Cap2 or Cap4). In some embodiments, polynucleotide comprising the Cap15′UTR, increases MCM expression levels in cells when introduced intothose cells, e.g., by at least 20%, e.g., at least 20%, at least 25%, atleast 35%, or at least 40%.

In some embodiments, at least one of the regions of linked nucleosidesof C can comprise a sequence of linked nucleosides that can function asa 3′ UTR. The sequence of linked nucleosides can be a natural orsynthetic 3′ UTR. As a non-limiting example, the chimeric polynucleotidecan encode MCM and the sequence of linked nucleosides of C can encodethe native 3′ UTR of MCM or a non-heterologous 3′ UTR such as, but notlimited to a synthetic UTR.

In some embodiments, at least one of the regions of linked nucleosidesof A comprises a sequence of linked nucleosides that functions as a 5′UTR and at least one of the regions of linked nucleosides of C comprisesa sequence of linked nucleosides that functions as a 3′ UTR. In someembodiments, the 5′ UTR and the 3′ UTR can be from the same or differentspecies. In another embodiment, the 5′ UTR and the 3′ UTR can encode thenative untranslated regions from different proteins from the same ordifferent species.

Chimeric polynucleotides, including the parts or regions thereof, of thepresent disclosure can be classified as hemimers, gapmers, wingmers, orblockmers.

As used herein, a “hemimer” is a chimeric polynucleotide comprising aregion or part that comprises half of one pattern, percent, position orpopulation of a chemical modification(s) and half of a second pattern,percent, position or population of a chemical modification(s). Chimericpolynucleotides of the present disclosure can also comprise hemimersubregions. In some embodiments, a part or region is 50% of one and 50%of another.

In some embodiments, the entire chimeric polynucleotide can be 50% ofone and 50% of the other. Any region or part of any chimericpolynucleotide of the disclosure can be a hemimer. Types of hemimersinclude pattern hemimers, population hemimers or position hemimers. Bydefinition, hemimers are 50:50 percent hemimers.

As used herein, a “gapmer” is a chimeric polynucleotide having at leastthree parts or regions with a gap between the parts or regions. The“gap” can comprise a region of linked nucleosides or a single nucleosidethat differs from the chimeric nature of the two parts or regionsflanking it. The two parts or regions of a gapmer can be the same ordifferent from each other.

As used herein, a “wingmer” is a chimeric polynucleotide having at leastthree parts or regions with a gap between the parts or regions. Unlike agapmer, the two flanking parts or regions surrounding the gap in awingmer are the same in degree or kind. Such similarity can be in thelength of number of units of different modifications or in the number ofmodifications. The wings of a wingmer can be longer or shorter than thegap. The wing parts or regions can be 20, 30, 40, 50, 60 70, 80, 90 or95% greater or shorter in length than the region that comprises the gap.

As used herein, a “blockmer” is a patterned polynucleotide where partsor regions are of equivalent size or number and type of modifications.Regions or subregions in a blockmer can be 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248,249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276,277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 310, 320, 330, 340,350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480,490 or 500, nucleosides long.

Chimeric polynucleotides, including the parts or regions thereof, of thepresent disclosure having a chemical modification pattern are referredto as “pattern chimeras.” Pattern chimeras can also be referred to asblockmers. Pattern chimeras are those polynucleotides having a patternof modifications within, across or among regions or parts.

Patterns of modifications within a part or region are those that startand stop within a defined region. Patterns of modifications across apart or region are those patterns that start in on part or region andend in another adjacent part or region. Patterns of modifications amongparts or regions are those that begin and end in one part or region andare repeated in a different part or region, which is not necessarilyadjacent to the first region or part.

The regions or subregions of pattern chimeras or blockmers can havesimple alternating patterns such as ABAB[AB]n where each “A” and each“B” represent different chemical modifications (at least one of thebase, sugar or backbone linker), different types of chemicalmodifications (e.g., naturally occurring and non-naturally occurring),different percentages of modifications or different populations ofmodifications. The pattern can repeat n number of times where n=3-300.Further, each A or B can represent from 1-2500 units (e.g., nucleosides)in the pattern. Patterns can also be alternating multiples such asAABBAABB[AABB]n (an alternating double multiple) orAAABBBAAABBB[AAABBB]n (an alternating triple multiple) pattern. Thepattern can repeat n number of times where n=3-300.

Different patterns can also be mixed together to form a second orderpattern. For example, a single alternating pattern can be combined witha triple alternating pattern to form a second order alternating patternA′B′. One example would be [ABABAB][AAABBBAAABBB][ABABAB][AAABBBAAABBB][ABABAB][AAABBBAAABBB], where [ABABAB] is A′ and [AAABBBAAABBB] is B′.In like fashion, these patterns can be repeated n number of times, wheren=3-300.

Patterns can include three or more different modifications to form anABCABC[ABC]n pattern. These three component patterns can also bemultiples, such as AABBCCAABBCC[AABBCC]n and can be designed ascombinations with other patterns such as ABCABCAABBCCABCABCAABBCC, andcan be higher order patterns.

Regions or subregions of position, percent, and population modificationsneed not reflect an equal contribution from each modification type. Theycan form series such as “1-2-3-4,” “1-2-4-8,” where each integerrepresents the number of units of a particular modification type.Alternatively, they can be odd only, such as ‘1-3-3-1-3-1-5” or evenonly “2-4-2-4-6-4-8” or a mixture of both odd and even number of unitssuch as “1-3-4-2-5-7-3-3-4”.

Pattern chimeras can vary in their chemical modification by degree (suchas those described above) or by kind (e.g., different modifications).

Chimeric polynucleotides, including the parts or regions thereof, of thepresent disclosure having at least one region with two or more differentchemical modifications of two or more nucleoside members of the samenucleoside type (A, C, G, T, or U) are referred to as “positionallymodified” chimeras. Positionally modified chimeras are also referred toherein as “selective placement” chimeras or “selective placementpolynucleotides”. As the name implies, selective placement refers to thedesign of polynucleotides that, unlike polynucleotides in the art wherethe modification to any A, C, G, T or U is the same by virtue of themethod of synthesis, can have different modifications to the individualAs, Cs, Gs, Ts or Us in a polynucleotide or region thereof. For example,in a positionally modified chimeric polynucleotide, there can be two ormore different chemical modifications to any of the nucleoside types ofAs, Cs, Gs, Ts, or Us. There can also be combinations of two or more toany two or more of the same nucleoside type. For example, a positionallymodified or selective placement chimeric polynucleotide can comprise 3different modifications to the population of adenines in the moleculeand also have 3 different modifications to the population of cytosinesin the construct—all of which can have a unique, non-random, placement.

Chimeric polynucleotides, including the parts or regions thereof, of thepresent disclosure having a chemical modification percent are referredto as “percent chimeras.” Percent chimeras can have regions or partsthat comprise at least 1%, at least 2%, at least 5%, at least 8%, atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, or atleast 99% positional, pattern or population of modifications.Alternatively, the percent chimera can be completely modified as tomodification position, pattern, or population. The percent ofmodification of a percent chimera can be split between naturallyoccurring and non-naturally occurring modifications.

Chimeric polynucleotides, including the parts or regions thereof, of thepresent disclosure having a chemical modification population arereferred to as “population chimeras.” A population chimera can comprisea region or part where nucleosides (their base, sugar or backbonelinkage, or combination thereof) have a select population ofmodifications. Such modifications can be selected from functionalpopulations such as modifications that induce, alter or modulate aphenotypic outcome. For example, a functional population can be apopulation or selection of chemical modifications that increase thelevel of a cytokine. Other functional populations can individually orcollectively function to decrease the level of one or more cytokines.Use of a selection of these like-function modifications in a chimericpolynucleotide would therefore constitute a “functional populationchimera.” As used herein, a “functional population chimera” can be onewhose unique functional feature is defined by the population ofmodifications as described above or the term can apply to the overallfunction of the chimeric polynucleotide itself. For example, as a wholethe chimeric polynucleotide can function in a different or superior wayas compared to an unmodified or non-chimeric polynucleotide.

It should be noted that polynucleotides that have a uniform chemicalmodification of all of any of the same nucleoside type or a populationof modifications produced by mere downward titration of the samestarting modification in all of any of the same nucleoside type, or ameasured percent of a chemical modification of all any of the samenucleoside type but with random incorporation, such as where alluridines are replaced by a uridine analog, e.g., pseudouridine or5-methoxyuridine, are not considered chimeric polynucleotides. Likewise,polynucleotides having a uniform chemical modification of two, three, orfour of the same nucleoside type throughout the entire polynucleotide(such as all uridines and all cytosines, etc. are modified in the sameway) are not considered chimeric polynucleotides. One example of apolynucleotide that is not chimeric is the canonicalpseudouridine/5-methyl cytosine modified polynucleotide. These uniformpolynucleotides are arrived at entirely via in vitro transcription (IVT)enzymatic synthesis; and due to the limitations of the synthesizingenzymes, they contain only one kind of modification at the occurrence ofeach of the same nucleoside type, i.e., adenosine (A), thymidine (T),guanosine (G), cytidine (C) or uridine (U), found in the polynucleotide.Such polynucleotides can be characterized as IVT polynucleotides.

The chimeric polynucleotides of the present disclosure can bestructurally modified or chemically modified. When the chimericpolynucleotides of the present disclosure are chemically and/orstructurally modified, the polynucleotides can be referred to as“modified chimeric polynucleotides.”

In some embodiments, the chimeric polynucleotides can encode two or morepeptides or polypeptides of interest. Such peptides or polypeptides ofinterest include an enzyme and its substrate, a label and its bindingmolecule, a second messenger and its enzyme, or the components ofmultimeric proteins or complexes.

The regions or parts of the chimeric polynucleotides can be separated bya linker or spacer moiety. Such linkers or spaces can be nucleic acidbased or non-nucleosidic.

In some embodiments, the chimeric polynucleotides can include a sequenceencoding a self-cleaving peptide described herein, such as, but notlimited to, a 2A peptide. The polynucleotide sequence of the 2A peptidein the chimeric polynucleotide can be modified or sequence-optimized bythe methods described herein and/or are known in the art.

Notwithstanding the foregoing, the chimeric polynucleotides of thepresent disclosure can comprise a region or part that is notpositionally modified or not chimeric as defined herein. For example, aregion or part of a chimeric polynucleotide can be uniformly modified atone or more A, T, C, G, or U, but the polynucleotides will not beuniformly modified throughout the entire region or part.

Regions or parts of chimeric polynucleotides can be, in someembodiments, from 15-10,000 nucleosides in length and, in someembodiments, a polynucleotide can have from 2-100 different regions orpatterns of regions as described herein.

In some embodiments, chimeric polynucleotides encode one or morepolypeptides of interest. In another embodiment, the chimericpolynucleotides are substantially non-coding. In another embodiment, thechimeric polynucleotides have both coding and non-coding regions andparts.

In some embodiments, regions or subregions of the polynucleotides canrange from being absent to 500 nucleotides in length (e.g., at least 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250,300, 350, 400, 450, or 500 nucleotides). Where the region is a polyAtail, the length can be determined in units of, or as a function of,polyA Binding Protein binding. In this embodiment, the polyA tail islong enough to bind at least 4 monomers of PolyA Binding Protein. PolyABinding Protein monomers bind to stretches of approximately 38nucleotides. As such, it has been observed that polyA tails of about 80nucleotides to about 160 nucleotides are functional. The chimericpolynucleotides of the present disclosure that function as an mRNA neednot comprise a polyA tail.

In some embodiments of the present disclosure, chimeric polynucleotidesthat function as an mRNA have a capping region. The capping region cancomprise a single cap or a series of nucleotides forming the cap. Inthis embodiment the capping region can be from 1 to 10, e.g., 2-9, 3-8,4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length. Insome embodiments, the cap is absent.

The present disclosure contemplates chimeric polynucleotides that arecircular or cyclic. As the name implies circular polynucleotides arecircular in nature meaning that the termini are joined in some fashion,whether by ligation, covalent bond, common association with the sameprotein or other molecule or complex or by hybridization.

Chimeric polynucleotides, formulations and compositions comprisingchimeric polynucleotides, and methods of making, using and administeringchimeric polynucleotides are also described in International PatentApplication No. PCT/US2014/53907, the contents of which is incorporatedby reference in its entirety.

In some embodiments, the chimeric polynucleotide encodes an MCM proteinor a functional fragment thereof. In some embodiments, the chimericpolynucleotides of the disclosure comprise any one of the human MCMnucleic acid sequences selected from SEQ ID NOs: 1-207, 732-765, and772. In some embodiments, the chimeric polynucleotide encodes a humanMCM or functional fragment thereof comprising at least one amino acidmutation from the wild type sequence. In some embodiments, the chimericpolynucleotide encodes an MCM mutant comprising one or more of the pointmutations V69, T499, H532, A598, and V671. In some embodiments, theexpression of the encoded polypeptide is increased. In some embodiments,the chimeric polynucleotide increases MCM expression levels in cellswhen introduced into those cells, e.g., by 20-50%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, or atleast 50%.

Circular Polynucleotide Architecture

The disclosure also includes a circular polynucleotide comprising thepolynucleotide described herein, i.e., a polynucleotide comprising anORF encoding an MCM polypeptide.

Polynucleotides that are circular are known as “circularpolynucleotides” or “circP.” As used herein, “circular polynucleotides”or “circP” means a single stranded circular polynucleotide which actssubstantially like, and has the properties of, an RNA. The term“circular” is also meant to encompass any secondary or tertiaryconfiguration of the circP.

The present disclosure contemplates polynucleotides encoding MCM thatare circular or cyclic. As the name implies circular polynucleotides arecircular in nature meaning that the termini are joined in some fashion,whether by ligation, covalent bond, common association with the sameprotein or other molecule or complex or by hybridization.

The circular polynucleotides or circPs that encode at least one peptideor polypeptide of interest are known as circular RNAs or circRNA. Asused herein, “circular RNA” or “circRNA” means a circular polynucleotidethat can encode at least one peptide or polypeptide of interest.

The circPs that comprise at least one sensor sequence and do not encodea peptide or polypeptide of interest are known as circular sponges orcircSP. As used herein, “circular sponges,” “circular polynucleotidesponges” or “circSP” means a circular polynucleotide that comprises atleast one sensor sequence and does not encode a polypeptide of interest.

As used herein, “sensor sequence” means a receptor or pseudo-receptorfor endogenous nucleic acid binding molecules. Non-limiting examples ofsensor sequences include, microRNA binding sites, microRNA seedsequences, microRNA binding sites without the seed sequence,transcription factor binding sites and artificial binding sitesengineered to act as pseudo-receptors and portions and fragmentsthereof.

The circPs that comprise at least one sensor sequence and encode atleast one peptide or polypeptide of interest are known as circular RNAsponges or circRNA-SP. As used herein, “circular RNA sponges” or“circRNA-SP” means a circular polynucleotide that comprises at least onesensor sequence and at least one region encoding at least one peptide orpolypeptide of interest.

As used herein, the term “circular construct” refers to a circularpolynucleotide transcript that can act substantially similar to and haveproperties of a RNA molecule. In some embodiments, the circularconstruct acts as an mRNA. If the circular construct encodes one or morepeptides or polypeptides of interest (e.g., a circRNA or circRNA-SP)then the polynucleotide transcript retains sufficient structural and/orchemical features to allow the polypeptide of interest encoded thereinto be translated. Circular constructs can be polynucleotides of thedisclosure. When structurally or chemically modified, the construct canbe referred to as a modified circP, modified circSP, modified circRNA ormodified circRNA-SP.

Circular polynucleotides, formulations and compositions comprisingcircular polynucleotides, and methods of making, using and administeringcircular polynucleotides are also disclosed in International PatentApplication No. PCT/US2014/53904 the contents of which is incorporatedby reference in its entirety.

In some embodiments, the circular polynucleotide encodes an MCM proteinor a functional fragment thereof. In some embodiments, the circularpolynucleotides of the disclosure comprise any one of the human MCMnucleic acid selected from SEQ ID NOs: 1-207, 732-765, and 772. In someembodiments, the circular polynucleotide encodes a human MCM orfunctional fragment thereof comprising at least one amino acid mutationfrom the wild type sequence. In some embodiments, the circularpolynucleotide encodes an MCM mutant comprising one or more of the pointmutations V69, T499, H532, A598, and V671. In some embodiments, theexpression of the encoded polypeptide is increased. In some embodiments,the circular polynucleotide increases MCM expression levels in cellswhen introduced into those cells, e.g., by 20-50%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, or atleast 50%.

Multimers of Polynucleotides

The disclosure also includes multimers of polynucleotides comprising thepolynucleotide described herein, i.e., a polynucleotide comprising anORF encoding an MCM polypeptide.

In some embodiments, multiple distinct chimeric polynucleotides and/orIVT polynucleotides can be linked together through the 3′-end usingnucleotides that are modified at the 3′-terminus. Chemical conjugationcan be used to control the stoichiometry of delivery into cells. Forexample, the glyoxylate cycle enzymes, isocitrate lyase and malatesynthase, can be supplied into cells at a 1:1 ratio to alter cellularfatty acid metabolism. This ratio can be controlled by chemicallylinking chimeric polynucleotides and/or IVT polynucleotides using a3′-azido terminated nucleotide on one polynucleotides species and aC5-ethynyl or alkynyl-containing nucleotide on the oppositepolynucleotide species. The modified nucleotide is addedpost-transcriptionally using terminal transferase (New England Biolabs,Ipswich, Mass.) according to the manufacturer's protocol. After theaddition of the 3′-modified nucleotide, the two polynucleotides speciescan be combined in an aqueous solution, in the presence or absence ofcopper, to form a new covalent linkage via a click chemistry mechanismas described in the literature.

In another example, more than two chimeric polynucleotides and/or IVTpolynucleotides can be linked together using a functionalized linkermolecule. For example, a functionalized saccharide molecule can bechemically modified to contain multiple chemical reactive groups (SH—,NH₂—, N₃, etc. . . . ) to react with the cognate moiety on a3′-functionalized mRNA molecule (i.e., a 3′-maleimide ester,3′-NHS-ester, alkynyl). The number of reactive groups on the modifiedsaccharide can be controlled in a stoichiometric fashion to directlycontrol the stoichiometric ratio of conjugated chimeric polynucleotidesand/or IVT polynucleotides.

In some embodiments, the chimeric polynucleotides and/or IVTpolynucleotides can be linked together in a pattern. The pattern can bea simple alternating pattern such as CD[CD]_(x) where each “C” and each“D” represent a chimeric polynucleotide, IVT polynucleotide, differentchimeric polynucleotides or different IVT polynucleotides. The patterncan repeat x number of times, where x=1-300. Patterns can also bealternating multiples such as CCDD[CCDD]_(x) (an alternating doublemultiple) or CCCDDD[CCCDDD]_(x) (an alternating triple multiple)pattern. The alternating double multiple or alternating triple multiplecan repeat x number of times, where x=1-300.

Conjugates and Combinations of Polynucleotides

The disclosure also includes conjugates and combinations ofpolynucleotides comprising the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide.

In order to further enhance protein production, polynucleotides of thepresent disclosure can be designed to be conjugated to otherpolynucleotides, dyes, or other agents.

Conjugation can result in increased stability and/or half-life and canbe particularly useful in targeting the polynucleotides to specificsites in the cell, tissue or organism.

In some embodiments, the polynucleotides can be administered with,conjugated to or further encode one or more of RNAi agents, siRNAs,shRNAs, miRNAs, miRNA binding sites, antisense RNAs, ribozymes,catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamersor vectors, and the like.

Bifunctional Polynucleotides

The disclosure also includes bifunctional polynucleotides comprising thepolynucleotide described herein, i.e., a polynucleotide comprising anORF encoding an MCM polypeptide.

In some embodiments of the disclosure are bifunctional polynucleotides(e.g., bifunctional IVT polynucleotides, bifunctional chimericpolynucleotides or bifunctional circular polynucleotides). As the nameimplies, bifunctional polynucleotides are those having or capable of atleast two functions. These molecules are also by convention be referredto as multi-functional.

The multiple functionalities of bifunctional polynucleotides can beencoded by the RNA (the function cannot manifest until the encodedproduct is translated) or can be a property of the polynucleotideitself. It can be structural or chemical. Bifunctional modifiedpolynucleotides can comprise a function that is covalently orelectrostatically associated with the polynucleotides. Further, the twofunctions can be provided in the context of a complex of a chimericpolynucleotide and another molecule.

Bifunctional polynucleotides can encode peptides that areanti-proliferative. These peptides can be linear, cyclic, constrained orrandom coil. They can function as aptamers, signaling molecules, ligandsor mimics or mimetics thereof. Anti-proliferative peptides can, astranslated, be from 3 to 50 amino acids in length. They can be 5-40,10-30, or approximately 15 amino acids long. They can be single chain,multichain or branched and can form complexes, aggregates or anymulti-unit structure once translated.

Noncoding Polynucleotides

The disclosure also includes a noncoding polynucleotide comprising thepolynucleotide described herein, i.e., a polynucleotide comprising anORF encoding an MCM polypeptide.

The polynucleotides described herein can further comprise sequences thatare partially or substantially not translatable, e.g., having anoncoding region. As one non-limiting example, the noncoding region canbe the first region of the IVT polynucleotide or the circularpolynucleotide. Alternatively, the noncoding region can be a regionother than the first region. As another non-limiting example, thenoncoding region can be the A, B and/or C region of the chimericpolynucleotide.

Such molecules are generally not translated, but can exert an effect onprotein production by one or more of binding to and sequestering one ormore translational machinery components such as a ribosomal protein or atransfer RNA (tRNA), thereby effectively reducing protein expression inthe cell or modulating one or more pathways or cascades in a cell thatin turn alters protein levels. The polynucleotide can contain or encodeone or more long noncoding RNA (lncRNA, or lincRNA) or portion thereof,a small nucleolar RNA (sno-RNA), micro RNA (miRNA), small interferingRNA (siRNA) or Piwi-interacting RNA (piRNA). Examples of such lncRNAmolecules and RNAi constructs designed to target such lncRNA any ofwhich can be encoded in the polynucleotides are disclosed inInternational Publication, WO2012/018881 A2, the contents of which areincorporated herein by reference in their entirety.

Cytotoxic Nucleosides

In some embodiments, the polynucleotides of the present disclosure(i.e., a polynucleotide comprising an ORF encoding an MCM polypeptide)can further incorporate one or more cytotoxic nucleosides.

Untranslated Regions (UTRs)

Untranslated regions (UTRs) are nucleic acid sections of apolynucleotide before a start codon (5′UTR) and after a stop codon(3′UTR) that are not translated. In some embodiments, a polynucleotide(e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)) of thedisclosure comprising an open reading frame (ORF) encoding an MCMpolypeptide further comprises UTR (e.g., a 5′UTR or functional fragmentthereof, a 3′UTR or functional fragment thereof, or a combinationthereof).

A UTR can be homologous or heterologous to the coding region in apolynucleotide. In some embodiments, the UTR is homologous to the ORFencoding the MCM polypeptide. In some embodiments, the UTR isheterologous to the ORF encoding the MCM polypeptide. In someembodiments, the polynucleotide comprises two or more 5′UTRs orfunctional fragments thereof, each of which have the same or differentnucleotide sequences. In some embodiments, the polynucleotide comprisestwo or more 3′UTRs or functional fragments thereof, each of which havethe same or different nucleotide sequences.

In some embodiments, the 5′UTR or functional fragment thereof, 3′ UTR orfunctional fragment thereof, or any combination thereof is sequenceoptimized.

In some embodiments, the 5′UTR or functional fragment thereof, 3′ UTR orfunctional fragment thereof, or any combination thereof comprises atleast one chemically modified nucleobase, e.g., 5-methoxyuracil.

UTRs can have features that provide a regulatory role, e.g., increasedor decreased stability, localization and/or translation efficiency. Apolynucleotide comprising a UTR can be administered to a cell, tissue,or organism, and one or more regulatory features can be measured usingroutine methods. In some embodiments, a functional fragment of a 5′UTRor 3′UTR comprises one or more regulatory features of a full length 5′or 3′ UTR, respectively.

Natural 5′UTRs bear features that play roles in translation initiation.They harbor signatures like Kozak sequences that are commonly known tobe involved in the process by which the ribosome initiates translationof many genes. Kozak sequences have the consensus CCR(A/G)CCAUGG, whereR is a purine (adenine or guanine) three bases upstream of the startcodon (AUG), which is followed by another ‘G’. 5′UTRs also have beenknown to form secondary structures that are involved in elongationfactor binding.

By engineering the features typically found in abundantly expressedgenes of specific target organs, one can enhance the stability andprotein production of a polynucleotide. For example, introduction of5′UTR of liver-expressed mRNA, such as albumin, serum amyloid A,Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, orFactor VIII, can enhance expression of polynucleotides in hepatic celllines or liver. Likewise, use of 5′UTR from other tissue-specific mRNAto improve expression in that tissue is possible for muscle (e.g., MyoD,Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (e.g.,Tie-1, CD36), for myeloid cells (e.g., C/EBP, AML1, G-CSF, GM-CSF,CD11b, MSR, Fr-1, i-NOS), for leukocytes (e.g., CD45, CD18), for adiposetissue (e.g., CD36, GLUT4, ACRP30, adiponectin) and for lung epithelialcells (e.g., SP-A/B/C/D).

In some embodiments, UTRs are selected from a family of transcriptswhose proteins share a common function, structure, feature or property.For example, an encoded polypeptide can belong to a family of proteins(i.e., that share at least one function, structure, feature,localization, origin, or expression pattern), which are expressed in aparticular cell, tissue or at some time during development. The UTRsfrom any of the genes or mRNA can be swapped for any other UTR of thesame or different family of proteins to create a new polynucleotide.

In some embodiments, the 5′UTR and the 3′UTR can be heterologous. Insome embodiments, the 5′UTR can be derived from a different species thanthe 3′UTR. In some embodiments, the 3′UTR can be derived from adifferent species than the 5′UTR.

Co-owned International Patent Application No. PCT/US2014/021522 (Publ.No. WO/2014/164253, incorporated herein by reference in its entirety)provides a listing of exemplary UTRs that can be utilized in thepolynucleotide of the present disclosure as flanking regions to an ORF.

Exemplary UTRs of the application include, but are not limited to, oneor more 5′UTR and/or 3′UTR derived from the nucleic acid sequence of: aglobin, such as an α- or β-globin (e.g., a Xenopus, mouse, rabbit, orhuman globin); a strong Kozak translational initiation signal; a CYBA(e.g., human cytochrome b-245α polypeptide); an albumin (e.g., humanalbumin7); a HSD17B4 (hydroxysteroid (17-β) dehydrogenase); a virus(e.g., a tobacco etch virus (TEV), a Venezuelan equine encephalitisvirus (VEEV), a Dengue virus, a cytomegalovirus (CMV) (e.g., CMVimmediate early 1 (IE1)), a hepatitis virus (e.g., hepatitis B virus), asindbis virus, or a PAV barley yellow dwarf virus); a heat shock protein(e.g., hsp70); a translation initiation factor (e.g., elF4G); a glucosetransporter (e.g., hGLUT1 (human glucose transporter 1)); an actin(e.g., human α or β actin); a GAPDH; a tubulin; a histone; a citric acidcycle enzyme; a topoisomerase (e.g., a 5′UTR of a TOP gene lacking the5′ TOP motif (the oligopyrimidine tract)); a ribosomal protein Large 32(L32); a ribosomal protein (e.g., human or mouse ribosomal protein, suchas, for example, rps9); an ATP synthase (e.g., ATP5A1 or the 3 subunitof mitochondrial H⁺-ATP synthase); a growth hormone e (e.g., bovine(bGH) or human (hGH)); an elongation factor (e.g., elongation factor 1α1 (EEF1A1)); a manganese superoxide dismutase (MnSOD); a myocyteenhancer factor 2A (MEF2A); a β-F1-ATPase, a creatine kinase, amyoglobin, a granulocyte-colony stimulating factor (G-CSF); a collagen(e.g., collagen type I, alpha 2 (Col1A2), collagen type I, alpha 1(Col1A1), collagen type VI, alpha 2 (Col6A2), collagen type VI, alpha 1(Col6A1)); a ribophorin (e.g., ribophorin I (RPNI)); a low densitylipoprotein receptor-related protein (e.g., LRP1); a cardiotrophin-likecytokine factor (e.g., Nntl); calreticulin (Calr); a procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (Plod1); and a nucleobindin (e.g., Nucb1).

Other exemplary 5′ and 3′ UTRs include, but are not limited to, thosedescribed in Karikó et al., Mol. Ther. 2008 16(11):1833-1840; Karikó etal., Mol. Ther. 2012 20(5):948-953; Karikó et al., Nucleic Acids Res.2011 39(21):e142; Strong et al., Gene Therapy 1997 4:624-627; Hansson etal., J. Biol. Chem. 2015 290(9):5661-5672; Yu et al., Vaccine 200725(10):1701-1711; Cafri et al., Mol. Ther. 2015 23(8):1391-1400; Andrieset al., Mol. Pharm. 2012 9(8):2136-2145; Crowley et al., Gene Ther. 2015Jun. 30, doi:10.1038/gt.2015.68; Ramunas et al., FASEB J. 201529(5):1930-1939; Wang et al., Curr. Gene Ther. 2015 15(4):428-435;Holtkamp et al., Blood 2006 108(13):4009-4017; Kormann et al., Nat.Biotechnol. 2011 29(2):154-157; Poleganov et al., Hum. Gen. Ther. 201526(11):751-766; Warren et al., Cell Stem Cell 2010 7(5):618-630; Mandaland Rossi, Nat. Protoc. 2013 8(3):568-582; Holcik and Liebhaber, PNAS1997 94(6):2410-2414; Ferizi et al., Lab Chip. 2015 15(17):3561-3571;Thess et al., Mol. Ther. 2015 23(9):1456-1464; Boros et al., PLoS One2015 10(6):e0131141; Boros et al., J. Photochem. Photobiol. B. 2013129:93-99; Andries et al., J. Control. Release 2015 217:337-344;Zinckgraf et al., Vaccine 2003 21(15):1640-9; Garneau et al., J. Virol.2008 82(2):880-892; Holden and Harris, Virology 2004 329(1):119-133;Chiu et al., J. Virol. 2005 79(13):8303-8315; Wang et al., EMBO J. 199716(13):4107-4116; Al-Zoghaibi et al., Gene 2007 391(1-2):130-9; Vivinuset al., Eur. J. Biochem. 2001 268(7):1908-1917; Gan and Rhoads, J. Biol.Chem. 1996 271(2):623-626; Boado et al., J. Neurochem. 199667(4):1335-1343; Knirsch and Clerch, Biochem. Biophys. Res. Commun. 2000272(1):164-168; Chung et al., Biochemistry 1998 37(46):16298-16306;Izquierdo and Cuevza, Biochem. J. 2000 346 Pt 3:849-855; Dwyer et al.,J. Neurochem. 1996 66(2):449-458; Black et al., Mol. Cell. Biol. 199717(5):2756-2763; Izquierdo and Cuevza, Mol. Cell. Biol. 199717(9):5255-5268; U.S. Pat. No. 8,278,036; U.S. Pat. No. 8,748,089; U.S.Pat. No. 8,835,108; U.S. Pat. No. 9,012,219; US2010/0129877;US2011/0065103; US2011/0086904; US2012/0195936; US2014/020675;US2013/0195967; US2014/029490; US2014/0206753; WO2007/036366;WO2011/015347; WO2012/072096; WO2013/143555; WO2014/071963;WO2013/185067; WO2013/182623; WO2014/089486; WO2013/185069;WO2014/144196; WO2014/152659; 2014/152673; WO2014/152940; WO2014/152774;WO2014/153052; WO2014/152966, WO2014/152513; WO2015/101414;WO2015/101415; WO2015/062738; and WO2015/024667; the contents of each ofwhich are incorporated herein by reference in their entirety.

In some embodiments, the 5′UTR is selected from the group consisting ofa β-globin 5′UTR; a 5′UTR containing a strong Kozak translationalinitiation signal; a cytochrome b-245α polypeptide (CYBA) 5′UTR; ahydroxysteroid (17-β) dehydrogenase (HSD17B4) 5′UTR; a Tobacco etchvirus (TEV) 5′UTR; a Venezuelen equine encephalitis virus (TEEV) 5′UTR;a 5′ proximal open reading frame of rubella virus (RV) RNA encodingnonstructural proteins; a Dengue virus (DEN) 5′UTR; a heat shock protein70 (Hsp70) 5′UTR; a eIF4G 5′UTR; a GLUT1 5′UTR; functional fragmentsthereof and any combination thereof.

In some embodiments, the 3′UTR is selected from the group consisting ofa β-globin 3′UTR; a CYBA 3′UTR; an albumin 3′UTR; a growth hormone (GH)3′UTR; a VEEV 3′UTR; a hepatitis B virus (HBV) 3′UTR; α-globin 3′UTR; aDEN 3′UTR; a PAV barley yellow dwarf virus (BYDV-PAV) 3′UTR; anelongation factor 1 α1 (EEF1A1) 3′UTR; a manganese superoxide dismutase(MnSOD) 3′UTR; a β subunit of mitochondrial H(+)-ATP synthase (β-mRNA)3′UTR; a GLUT1 3′UTR; a MEF2A 3′UTR; a β-F1-ATPase 3′UTR; functionalfragments thereof and combinations thereof.

Other exemplary UTRs include, but are not limited to, one or more of theUTRs, including any combination of UTRs, disclosed in WO2014/164253, thecontents of which are incorporated herein by reference in theirentirety. Shown in Table 21 of U.S. Provisional Application No.61/775,509 and in Table 22 of U.S. Provisional Application No.61/829,372, the contents of each are incorporated herein by reference intheir entirety, is a listing start and stop sites for 5′UTRs and 3′UTRs.In Table 21, each 5′UTR (5′-UTR-005 to 5′-UTR 68511) is identified byits start and stop site relative to its native or wild-type (homologous)transcript (ENST; the identifier used in the ENSEMBL database).

Wild-type UTRs derived from any gene or mRNA can be incorporated intothe polynucleotides of the disclosure. In some embodiments, a UTR can bealtered relative to a wild type or native UTR to produce a variant UTR,e.g., by changing the orientation or location of the UTR relative to theORF; or by inclusion of additional nucleotides, deletion of nucleotides,swapping or transposition of nucleotides. In some embodiments, variantsof 5′ or 3′ UTRs can be utilized, for example, mutants of wild typeUTRs, or variants wherein one or more nucleotides are added to orremoved from a terminus of the UTR.

Additionally, one or more synthetic UTRs can be used in combination withone or more non-synthetic UTRs. See, e.g., Mandal and Rossi, Nat.Protoc. 2013 8(3):568-82, the contents of which are incorporated hereinby reference in their entirety, and sequences available atwww.addgene.org/Derrick_Rossi/. UTRs or portions thereof can be placedin the same orientation as in the transcript from which they wereselected or can be altered in orientation or location. Hence, a 5′and/or 3′ UTR can be inverted, shortened, lengthened, or combined withone or more other 5′ UTRs or 3′ UTRs.

In some embodiments, the polynucleotide comprises multiple UTRs, e.g., adouble, a triple or a quadruple 5′UTR or 3′UTR. For example, a doubleUTR comprises two copies of the same UTR either in series orsubstantially in series. For example, a double beta-globin 3′UTR can beused (see US2010/0129877, the contents of which are incorporated hereinby reference in its entirety).

Tables 5 and 6 provide a listing of exemplary UTRs that can be utilizedin the polynucleotides of the present disclosure. Shown in Table 5 is alisting of a 5′-untranslated region of the disclosure. Variants of 5′UTRs can be utilized wherein one or more nucleotides are added orremoved to the termini, including A, T, C or G.

TABLE 5 5′-Untranslated Regions 5′ UTR Name/ SEQ ID IdentifierDescription Sequence NO. 5UTR-001 Upstream UTRGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATAT 215 AAGAGCCACC 5UTR-002Upstream UTR GGGAGATCAGAGAGAAAAGAAGAGTAAGAAGAAATAT 216 AAGAGCCACC5UTR-003 Upstream UTR GGAATAAAAGTCTCAACACAACATATACAAAACAAAC 217GAATCTCAAGCAATCAAGCATTCTACTTCTATTGCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTT CTGAAAATTTTCACCATTTACGAACGATAGCAAC5UTR-004 Upstream UTR GGGAGACAAGCUUGGCAUUCCGGUACUGUUGGUAAAG 218 CCACC5UTR-005 Upstream UTR GGGAGATCAGAGAGAAAAGAAGAGTAAGAAGAAATAT 219AAGAGCCACC 5UTR-006 Upstream UTR GGAATAAAAGTCTCAACACAACATATACAAAACAAAC220 GAATCTCAAGCAATCAAGCATTCTACTTCTATTGCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTT CTGAAAATTTTCACCATTTACGAACGATAGCAAC5UTR-007 Upstream UTR GGGAGACAAGCUUGGCAUUCCGGUACUGUUGGUAAAG 221 CCACC5UTR-008 Upstream UTR GGGAATTAACAGAGAAAAGAAGAGTAAGAAGAAATAT 222AAGAGCCACC 5UTR-009 Upstream UTR GGGAAATTAGACAGAAAAGAAGAGTAAGAAGAAATAT223 AAGAGCCACC 5UTR-010 Upstream UTRGGGAAATAAGAGAGTAAAGAACAGTAAGAAGAAATAT 224 AAGAGCCACC 5UTR-011Upstream UTR GGGAAAAAAGAGAGAAAAGAAGACTAAGAAGAAATAT 225 AAGAGCCACC5UTR-012 Upstream UTR GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGATATAT 226AAGAGCCACC 5UTR-013 Upstream UTR GGGAAATAAGAGACAAAACAAGAGTAAGAAGAAATAT227 AAGAGCCACC 5UTR-014 Upstream UTRGGGAAATTAGAGAGTAAAGAACAGTAAGTAGAATTAA 228 AAGAGCCACC 5UTR-015Upstream UTR GGGAAATAAGAGAGAATAGAAGAGTAAGAAGAAATAT 229 AAGAGCCACC5UTR-016 Upstream UTR GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAAATT 230AAGAGCCACC 5UTR-017 Upstream UTR GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATTT231 AAGAGCCACC 5UTR-018 Upstream UTRTCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGAC 266TCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGA AGAAATATAAGAGCCACC 142-3pUpstream UTR TGATAATAGTCCATAAAGTAGGAAACACTACAGCTGG 725 UTR-001 includingAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCC miR142-3pCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCC GTGGTCTTTGAATAAAGTCTGAGTGGGCGGC142-3p Upstream UTR TGATAATAGGCTGGAGCCTCGGTGGCTCCATAAAGTA 726 UTR-002including GGAAACACTACACATGCTTCTTGCCCCTTGGGCCTCC miR142-3pCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCC GTGGTCTTTGAATAAAGTCTGAGTGGGCGGC142-3p Upstream UTR TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTG 727 UTR-003including CCCCTTCCATAAAGTAGGAAACACTACATGGGCCTCC miR142-3pCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCC GTGGTCTTTGAATAAAGTCTGAGTGGGCGGC142-3p Upstream UTR TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTG 728 UTR-004including CCCCTTGGGCCTCCCCCCAGTCCATAAAGTAGGAAAC miR142-3pACTACACCCCTCCTCCCCTTCCTGCACCCGTACCCCC GTGGTCTTTGAATAAAGTCTGAGTGGGCGGC142-3p Upstream UTR TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTG 729 UTR-005including CCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCTC miR142-3pCATAAAGTAGGAAACACTACACTGCACCCGTACCCCC GTGGTCTTTGAATAAAGTCTGAGTGGGCGGC142-3p Upstream UTR TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTG 730 UTR-006including CCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCT miR142-3pGCACCCGTACCCCCTCCATAAAGTAGGAAACACTACA GTGGTCTTTGAATAAAGTCTGAGTGGGCGGC142-3p Upstream UTR TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTG 731 UTR-007including CCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCT miR142-3pGCACCCGTACCCCCGTGGTCTTTGAATAAAGTTCCAT AAAGTAGGAAACACTACACTGAGTGGGCGGC

In a particular embodiment, the 5′ UTR useful for the polynucleotidescomprises SEQ ID NO: 266.

Shown in Table 6 is a listing of 3′-untranslated regions of thedisclosure. Variants of 3′ UTRs can be utilized wherein one or morenucleotides are added or removed to the termini, including A, T, C or G.

TABLE 6 3′-Untranslated Regions 3′ UTR Name/ SEQ ID IdentifierDescription Sequence NO. 3UTR-001 CreatineGCGCCTGCCCACCTGCCACCGACTGCTGGAACCCAGCCAGTGG 232 KinaseGAGGGCCTGGCCCACCAGAGTCCTGCTCCCTCACTCCTCGCCCCGCCCCCTGTCCCAGAGTCCCACCTGGGGGCTCTCTCCACCCTTCTCAGAGTTCCAGTTTCAACCAGAGTTCCAACCAATGGGCTCCATCCTCTGGATTCTGGCCAATGAAATATCTCCCTGGCAGGGTCCTCTTCTTTTCCCAGAGCTCCACCCCAACCAGGAGCTCTAGTTAATGGAGAGCTCCCAGCACACTCGGAGCTTGTGCTTTGTCTCCACGCAAAGCGATAAATAAAAGCATTGGTGGCCTTTGGTCTTT GAATAAAGCCTGAGTAGGAAGTCTAGA3UTR-002 Myoglobin GCCCCTGCCGCTCCCACCCCCACCCATCTGGGCCCCGGGTTCA 233AGAGAGAGCGGGGTCTGATCTCGTGTAGCCATATAGAGTTTGCTTCTGAGTGTCTGCTTTGTTTAGTAGAGGTGGGCAGGAGGAGCTGAGGGGCTGGGGCTGGGGTGTTGAAGTTGGCTTTGCATGCCCAGCGATGCGCCTCCCTGTGGGATGTCATCACCCTGGGAACCGGGAGTGGCCCTTGGCTCACTGTGTTCTGCATGGTTTGGATCTGAATTAATTGTCCTTTCTTCTAAATCCCAACCGAACTTCTTCCAACCTCCAAACTGGCTGTAACCCCAAATCCAAGCCATTAACTACACCTGACAGTAGCAATTGTCTGATTAATCACTGGCCCCTTGAAGACAGCAGAATGTCCCTTTGCAATGAGGAGGAGATCTGGGCTGGGCGGGCCAGCTGGGGAAGCATTTGACTATCTGGAACTTGTGTGTGCCTCCTCAGGTATGGCAGTGACTCACCTGGTTTTAATAAAACAACCTGCAACATCTCATGGTCTTTGAATAAAGCCTGAGTAGG AAGTCTAGA 3UTR-003 α-actinACACACTCCACCTCCAGCACGCGACTTCTCAGGACGACGAATC 234TTCTCAATGGGGGGGCGGCTGAGCTCCAGCCACCCCGCAGTCACTTTCTTTGTAACAACTTCCGTTGCTGCCATCGTAAACTGACACAGTGTTTATAACGTGTACATACATTAACTTATTACCTCATTTTGTTATTTTTCGAAACAAAGCCCTGTGGAAGAAAATGGAAAACTTGAAGAAGCATTAAAGTCATTCTGTTAAGCTGCGTAAATGGTCTTTGAATAAAGCCTGAGTAGGAAGTCTAGA 3UTR-004 AlbuminCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGA 235AAGAAAATGAAGATCAAAAGCTTATTCATCTGTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAATCTAATAGAGTGGTACAGCACTGTTATTTTTCAAAGATGTGTTGCTATCCTGAAAATTCTGTAGGTTCTGTGGAAGTTCCAGTGTTCTCTCTTATTCCACTTCGGTAGAGGATTTCTAGTTTCTTGTGGGCTAATTAAATAAATCATTAATACTCTTCTAATGGTCTTTGAATAAAGCCTGAGTAGGAAGTCTAGA 3UTR-005 α-globinGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCT 236CTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCATGCATCTAGA 3UTR-006 G-CSFGCCAAGCCCTCCCCATCCCATGTATTTATCTCTATTTAATATT 237TATGTCTATTTAAGCCTCATATTTAAAGACAGGGAAGAGCAGAACGGAGCCCCAGGCCTCTGTGTCCTTCCCTGCATTTCTGAGTTTCATTCTCCTGCCTGTAGCAGTGAGAAAAAGCTCCTGTCCTCCCATCCCCTGGACTGGGAGGTAGATAGGTAAATACCAAGTATTTATTACTATGACTGCTCCCCAGCCCTGGCTCTGCAATGGGCACTGGGATGAGCCGCTGTGAGCCCCTGGTCCTGAGGGTCCCCACCTGGGACCCTTGAGAGTATCAGGTCTCCCACGTGGGAGACAAGAAATCCCTGTTTAATATTTAAACAGCAGTGTTCCCCATCTGGGTCCTTGCACCCCTCACTCTGGCCTCAGCCGACTGCACAGCGGCCCCTGCATCCCCTTGGCTGTGAGGCCCCTGGACAAGCAGAGGTGGCCAGAGCTGGGAGGCATGGCCCTGGGGTCCCACGAATTTGCTGGGGAATCTCGTTTTTCTTCTTAAGACTTTTGGGACATGGTTTGACTCCCGAACATCACCGACGCGTCTCCTGTTTTTCTGGGTGGCCTCGGGACACCTGCCCTGCCCCCACGAGGGTCAGGACTGTGACTCTTTTTAGGGCCAGGCAGGTGCCTGGACATTTGCCTTGCTGGACGGGGACTGGGGATGTGGGAGGGAGCAGACAGGAGGAATCATGTCAGGCCTGTGTGTGAAAGGAAGCTCCACTGTCACCCTCCACCTCTTCACCCCCCACTCACCAGTGTCCCCTCCACTGTCACATTGTAACTGAACTTCAGGATAATAAAGTGTTTGCCTCCATGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCATGCAT CTAGA 3UTR-007 Col1a2;ACTCAATCTAAATTAAAAAAGAAAGAAATTTGAAAAAACTTTC 238 collagen,TCTTTGCCATTTCTTCTTCTTCTTTTTTAACTGAAAGCTGAAT type I, alphaCCTTCCATTTCTTCTGCACATCTACTTGCTTAAATTGTGGGCA 2AAAGAGAAAAAGAAGGATTGATCAGAGCATTGTGCAATACAGTTTCATTAACTCCTTCCCCCGCTCCCCCAAAAATTTGAATTTTTTTTTCAACACTCTTACACCTGTTATGGAAAATGTCAACCTTTGTAAGAAAACCAAAATAAAAATTGAAAAATAAAAACCATAAACATTTGCACCACTTGTGGCTTTTGAATATCTTCCACAGAGGGAAGTTTAAAACCCAAACTTCCAAAGGTTTAAACTACCTCAAAACACTTTCCCATGAGTGTGATCCACATTGTTAGGTGCTGACCTAGACAGAGATGAACTGAGGTCCTTGTTTTGTTTTGTTCATAATACAAAGGTGCTAATTAATAGTATTTCAGATACTTGAAGAATGTTGATGGTGCTAGAAGAATTTGAGAAGAAATACTCCTGTATTGAGTTGTATCGTGTGGTGTATTTTTTAAAAAATTTGATTTAGCATTCATATTTTCCATCTTATTCCCAATTAAAAGTATGCAGATTATTTGCCCAAATCTTCTTCAGATTCAGCATTTGTTCTTTGCCAGTCTCATTTTCATCTTCTTCCATGGTTCCACAGAAGCTTTGTTTCTTGGGCAAGCAGAAAAATTAAATTGTACCTATTTTGTATATGTGAGATGTTTAAATAAATTGTGAAAAAAATGAAATAAAGCATGTTTGG TTTTCCAAAAGAACATAT 3UTR-008Col6a2; CGCCGCCGCCCGGGCCCCGCAGTCGAGGGTCGTGAGCCCACCC 239 collagen,CGTCCATGGTGCTAAGCGGGCCCGGGTCCCACACGGCCAGCAC IYpe VI,CGCTGCTCACTCGGACGACGCCCTGGGCCTGCACCTCTCCAGC alpha 2TCCTCCCACGGGGTCCCCGTAGCCCCGGCCCCCGCCCAGCCCCAGGTCTCCCCAGGCCCTCCGCAGGCTGCCCGGCCTCCCTCCCCCTGCAGCCATCCCAAGGCTCCTGACCTACCTGGCCCCTGAGCTCTGGAGCAAGCCCTGACCCAATAAAGGCTTTGAACCCAT 3UTR-009 RPN1;GGGGCTAGAGCCCTCTCCGCACAGCGTGGAGACGGGGCAAGGA 240 ribophorin IGGGGGGTTATTAGGATTGGTGGTTTTGTTTTGCTTTGTTTAAAGCCGTGGGAAAATGGCACAACTTTACCTCTGTGGGAGATGCAACACTGAGAGCCAAGGGGTGGGAGTTGGGATAATTTTTATATAAAAGAAGTTTTTCCACTTTGAATTGCTAAAAGTGGCATTTTTCCTATGTGCAGTCACTCCTCTCATTTCTAAAATAGGGACGTGGCCAGGCACGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCAGGCGGCTCACGAGGTCAGGAGATCGAGACTATCCTGGCTAACACGGTAAAACCCTGTCTCTACTAAAAGTACAAAAAATTAGCTGGGCGTGGTGGTGGGCACCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAAAGGCATGAATCCAAGAGGCAGAGCTTGCAGTGAGCTGAGATCACGCCATTGCACTCCAGCCTGGGCAACAGTGTTAAGACTCTGTCTCAAATATAAATAAATAAATAAATAAATAAATAAATAAATAAAAATAAAGCGAGATGTTGCCCTCAAA 3UTR-010 LRP1; lowGGCCCTGCCCCGTCGGACTGCCCCCAGAAAGCCTCCTGCCCCC 241 densityTGCCAGTGAAGTCCTTCAGTGAGCCCCTCCCCAGCCAGCCCTT lipoproteinCCCTGGCCCCGCCGGATGTATAAATGTAAAAATGAAGGAATTA receptor-CATTTTATATGTGAGCGAGCAAGCCGGCAAGCGAGCACAGTAT relatedTATTTCTCCATCCCCTCCCTGCCTGCTCCTTGGCACCCCCATG protein 1CTGCCTTCAGGGAGACAGGCAGGGAGGGCTTGGGGCTGCACCTCCTACCCTCCCACCAGAACGCACCCCACTGGGAGAGCTGGTGGTGCAGCCTTCCCCTCCCTGTATAAGACACTTTGCCAAGGCTCTCCCCTCTCGCCCCATCCCTGCTTGCCCGCTCCCACAGCTTCCTGAGGGCTAATTCTGGGAAGGGAGAGTTCTTTGCTGCCCCTGTCTGGAAGACGTGGCTCTGGGTGAGGTAGGCGGGAAAGGATGGAGTGTTTTAGTTCTTGGGGGAGGCCACCCCAAACCCCAGCCCCAACTCCAGGGGCACCTATGAGATGGCCATGCTCAACCCCCCTCCCAGACAGGCCCTCCCTGTCTCCAGGGCCCCCACCGAGGTTCCCAGGGCTGGAGACTTCCTCTGGTAAACATTCCTCCAGCCTCCCCTCCCCTGGGGACGCCAAGGAGGTGGGCCACACCCAGGAAGGGAAAGCGGGCAGCCCCGTTTTGGGGACGTGAACGTTTTAATAATTTTTGCTGAATTCCTTTACAACTAAATAACACAGATATTGTTATA AATAAAATTGT 3UTR-011 Nnt1;ATATTAAGGATCAAGCTGTTAGCTAATAATGCCACCTCTGCAG 242 cardio-TTTTGGGAACAGGCAAATAAAGTATCAGTATACATGGTGATGT trophin-ACATCTGTAGCAAAGCTCTTGGAGAAAATGAAGACTGAAGAAA likeGCAAAGCAAAAACTGTATAGAGAGATTTTTCAAAAGCAGTAAT cytokineCCCTCAATTTTAAAAAAGGATTGAAAATTCTAAATGTCTTTCT factor 1GTGCATATTTTTTGTGTTAGGAATCAAAAGTATTTTATAAAAGGAGAAAGAACAGCCTCATTTTAGATGTAGTCCTGTTGGATTTTTTATGCCTCCTCAGTAACCAGAAATGTTTTAAAAAACTAAGTGTTTAGGATTTCAAGACAACATTATACATGGCTCTGAAATATCTGACACAATGTAAACATTGCAGGCACCTGCATTTTATGTTTTTTTTTTCAACAAATGTGACTAATTTGAAACTTTTATGAACTTCTGAGCTGTCCCCTTGCAATTCAACCGCAGTTTGAATTAATCATATCAAATCAGTTTTAATTTTTTAAATTGTACTTCAGAGTCTATATTTCAAGGGCACATTTTCTCACTACTATTTTAATACATTAAAGGACTAAATAATCTTTCAGAGATGCTGGAAACAAATCATTTGCTTTATATGTTTCATTAGAATACCAATGAAACATACAACTTGAAAATTAGTAATAGTATTTTTGAAGATCCCATTTCTAATTGGAGATCTCTTTAATTTCGATCAACTTATAATGTGTAGTACTATATTAAGTGCACTTGAGTGGAATTCAACATTTGACTAATAAAATGAGTTCATCATGTTGGCAAGTGATGTGGCAATTATCTCTGGTGACAAAAGAGTAAAATCAAATATTTCTGCCTGTTACAAATATCAAGGAAGACCTGCTACTATGAAATAGATGACATTAATCTGTCTTCACTGTTTATAATACGGATGGATTTTTTTTCAAATCAGTGTGTGTTTTGAGGTCTTATGTAATTGATGACATTTGAGAGAAATGGTGGCTTTTTTTAGCTACCTCTTTGTTCATTTAAGCACCAGTAAAGATCATGTCTTTTTATAGAAGTGTAGATTTTCTTTGTGACTTTGCTATCGTGCCTAAAGCTCTAAATATAGGTGAATGTGTGATGAATACTCAGATTATTTGTCTCTCTATATAATTAGTTTGGTACTAAGTTTCTCAAAAAATTATTAACACATGAAAGACAATCTCTAAACCAGAAAAAGAAGTAGTACAAATTTTGTTACTGTAATGCTCGCGTTTAGTGAGTTTAAAACACACAGTATCTTTTGGTTTTATAATCAGTTTCTATTTTGCTGTGCCTGAGATTAAGATCTGTGTATGTGTGTGTGTGTGTGTGTGCGTTTGTGTGTTAAAGCAGAAAAGACTTTTTTAAAAGTTTTAAGTGATAAATGCAATTTGTTAATTGATCTTAGATCACTAGTAAACTCAGGGCTGAATTATACCATGTATATTCTATTAGAAGAAAGTAAACACCATCTTTATTCCTGCCCTTTTTCTTCTCTCAAAGTAGTTGTAGTTATATCTAGAAAGAAGCAATTTTGATTTCTTGAAAAGGTAGTTCCTGCACTCAGTTTAAACTAAAAATAATCATACTTGGATTTTATTTATTTTTGTCATAGTAAAAATTTTAATTTATATATATTTTTATTTAGTATTATCTTATTCTTTGCTATTTGCCAATCCTTTGTCATCAATTGTGTTAAATGAATTGAAAATTCATGCCCTGTTCATTTTATTTTACTTTATTGGTTAGGATATTTAAAGGATTTTTGTATATATAATTTCTTAAATTAATATTCCAAAAGGTTAGTGGACTTAGATTATAAATTATGGCAAAAATCTAAAAACAACAAAAATGATTTTTATACATTCTATTTCATTATTCCTCTTTTTCCAATAAGTCATACAATTGGTAGATATGACTTATTTTATTTTTGTATTATTCACTATATCTTTATGATATTTAAGTATAAATAATTAAAAAAATTTATTGTACCTTATAGTCTGTCACCAAAAAAAAAAAATTATCTGTAGGTAGTGAAATGCTAATGTTGATTTGTCTTTAAGGGCTTGTTAACTATCCTTTATTTTCTCATTTGTCTTAAATTAGGAGTTTGTGTTTAAATTACTCATCTAAGCAAAAAATGTATATAAATCCCATTACTGGGTATATACCCAAAGGATTATAAATCATGCTGCTATAAAGACACATGCACACGTATGTTTATTGCAGCACTATTCACAATAGCAAAGACTTGGAACCAACCCAAATGTCCATCAATGATAGACTTGATTAAGAAAATGTGCACATATACACCATGGAATACTATGCAGCCATAAAAAAGGATGAGTTCATGTCCTTTGTAGGGACATGGATAAAGCTGGAAACCATCATTCTGAGCAAACTATTGCAAGGACAGAAAACCAAACACTGCATGTTCTCACTCATAGGTGGGAATTGAACAATGAGAACACTTGGACACAAGGTGGGGAACACCACACACCAGGGCCTGTCATGGGGTGGGGGGAGTGGGGAGGGATAGCATTAGGAGATATACCTAATGTAAATGATGAGTTAATGGGTGCAGCACACCAACATGGCACATGTATACATATGTAGCAAACCTGCACGTTGTGCACATGTACCCTAGAACTTAAAGTATAATTAAAAAAAAAAAGAAAACAGAAGCTATTTATAAAGAAGTTATTTGCTGAAATAAATGTGATCTTTCCCATTAAAAAAATAAAGAAATTTTGGGGTAAAAAAACACAATATATTGTATTCTTGAAAAATTCTAAGAGAGTGGATGTGAAGTGTTCTCACCACAAAAGTGATAACTAATTGAGGTAATGCACATATTAATTAGAAAGATTTTGTCATTCCACAATGTATATATACTTAAAAATATGTTATACACAATAAATACATACATTAAAAAATAAGTAAATGTA 3UTR-012 Col6a1;CCCACCCTGCACGCCGGCACCAAACCCTGTCCTCCCACCCCTC 243 collagen,CCCACTCATCACTAAACAGAGTAAAATGTGATGCGAATTTTCC type VI,CGACCAACCTGATTCGCTAGATTTTTTTTAAGGAAAAGCTTGG alpha 1AAAGCCAGGACACAACGCTGCTGCCTGCTTTGTGCAGGGTCCTCCGGGGCTCAGCCCTGAGTTGGCATCACCTGCGCAGGGCCCTCTGGGGCTCAGCCCTGAGCTAGTGTCACCTGCACAGGGCCCTCTGAGGCTCAGCCCTGAGCTGGCGTCACCTGTGCAGGGCCCTCTGGGGCTCAGCCCTGAGCTGGCCTCACCTGGGTTCCCCACCCCGGGCTCTCCTGCCCTGCCCTCCTGCCCGCCCTCCCTCCTGCCTGCGCAGCTCCTTCCCTAGGCACCTCTGTGCTGCATCCCACCAGCCTGAGCAAGACGCCCTCTCGGGGCCTGTGCCGCACTAGCCTCCCTCTCCTCTGTCCCCATAGCTGGTTTTTCCCACCAATCCTCACCTAACAGTTACTTTACAATTAAACTCAAAGCAAGCTCTTCTCCTCAGCTTGGGGCAGCCATTGGCCTCTGTCTCGTTTTGGGAAACCAAGGTCAGGAGGCCGTTGCAGACATAAATCTCGGCGACTCGGCCCCGTCTCCTGAGGGTCCTGCTGGTGACCGGCCTGGACCTTGGCCCTACAGCCCTGGAGGCCGCTGCTGACCAGCACTGACCCCGACCTCAGAGAGTACTCGCAGGGGCGCTGGCTGCACTCAAGACCCTCGAGATTAACGGTGCTAACCCCGTCTGCTCCTCCCTCCCGCAGAGACTGGGGCCTGGACTGGACATGAGAGCCCCTTGGTGCCACAGAGGGCTGTGTCTTACTAGAAACAACGCAAACCTCTCCTTCCTCAGAATAGTGATGTGTTCGACGTTTTATCAAAGGCCCCCTTTCTATGTTCATGTTAGTTTTGCTCCTTCTGTGTTTTTTTCTGAACCATATCCATGTTGCTGACTTTTCCAAATAAAGGTTTTCACTC CTCTC 3UTR-013 Calr;AGAGGCCTGCCTCCAGGGCTGGACTGAGGCCTGAGCGCTCCTG 244 calreticulinCCGCAGAGCTGGCCGCGCCAAATAATGTCTCTGTGAGACTCGAGAACTTTCATTTTTTTCCAGGCTGGTTCGGATTTGGGGTGGATTTTGGTTTTGTTCCCCTCCTCCACTCTCCCCCACCCCCTCCCCGCCCTTTTTTTTTTTTTTTTTTAAACTGGTATTTTATCTTTGATTCTCCTTCAGCCCTCACCCCTGGTTCTCATCTTTCTTGATCAACATCTTTTCTTGCCTCTGTCCCCTTCTCTCATCTCTTAGCTCCCCTCCAACCTGGGGGGCAGTGGTGTGGAGAAGCCACAGGCCTGAGATTTCATCTGCTCTCCTTCCTGGAGCCCAGAGGAGGGCAGCAGAAGGGGGTGGTGTCTCCAACCCCCCAGCACTGAGGAAGAACGGGGCTCTTCTCATTTCACCCCTCCCTTTCTCCCCTGCCCCCAGGACTGGGCCACTTCTGGGTGGGGCAGTGGGTCCCAGATTGGCTCACACTGAGAATGTAAGAACTACAAACAAAATTTCTATTAA ATTAAATTTTGTGTCTCC 3UTR-014Col1a1; CTCCCTCCATCCCAACCTGGCTCCCTCCCACCCAACCAACTTT 245 collagen,CCCCCCAACCCGGAAACAGACAAGCAACCCAAACTGAACCCCC type I,TCAAAAGCCAAAAAATGGGAGACAATTTCACATGGACTTTGGA alpha 1AAATATTTTTTTCCTTTGCATTCATCTCTCAAACTTAGTTTTTATCTTTGACCAACCGAACATGACCAAAAACCAAAAGTGCATTCAACCTTACCAAAAAAAAAAAGAATAAATGAATAAATAAATAACTTTTTAAAAAAGGAAGCTTGGTCCACTTGCTTGAAGACCCATGCGGGGGTAAGTCCCTTTCTGCCCGTTGGGCTTATGAAACCCCAATGCTGCCCTTTCTGCTCCTTTCTCCACACCCCCCTTGGGGCCTCCCCTCCACTCCTTCCCAAATCTGTCTCCCCAGAAGACACAGGAAACAATGTATTGTCTGCCCAGCAATCAAAGGCAATGCTCAAACACCCAAGTGGCCCCCACCCTCAGCCCGCTCCTGCCCGCCCAGCACCCCCAGGCCCTGGGGGACCTGGGGTTCTCAGACTGCCAAAGAAGCCTTGCCATCTGGCGCTCCCATGGCTCTTGCAACATCTCCCCTTCGTTTTTGAGGGGGTCATGCCGGGGGAGCCACCAGCCCCTCACTGGGTTCGGAGGAGAGTCAGGAAGGGCCACGACAAAGCAGAAACATCGGATTTGGGGAACGCGTGTCAATCCCTTGTGCCGCAGGGCTGGGCGGGAGAGACTGTTCTGTTCCTTGTGTAACTGTGTTGCTGAAAGACTACCTCGTTCTTGTCTTGATGTGTCACCGGGGCAACTGCCTGGGGGCGGGGATGGGGGCAGGGTGGAAGCGGCTCCCCATTTTATACCAAAGGTGCTACATCTATGTGATGGGTGGGGTGGGGAGGGAATCACTGGTGCTATAGAAATTGAGATGCCCCCCCAGGCCAGCAAATGTTCCTTTTTGTTCAAAGTCTATTTTTATTCCTTGATATTTTTCTTTTTTTTTTTTTTTTTTTGTGGATGGGGACTTGTGAATTTTTCTAAAGGTGCTATTTAACATGGGAGGAGAGCGTGTGCGGCTCCAGCCCAGCCCGCTGCTCACTTTCCACCCTCTCTCCACCTGCCTCTGGCTTCTCAGGCCTCTGCTCTCCGACCTCTCTCCTCTGAAACCCTCCTCCACAGCTGCAGCCCATCCTCCCGGCTCCCTCCTAGTCTGTCCTGCGTCCTCTGTCCCCGGGTTTCAGAGACAACTTCCCAAAGCACAAAGCAGTTTTTCCCCCTAGGGGTGGGAGGAAGCAAAAGACTCTGTACCTATTTTGTATGTGTATAATAATTTGAGATGTTTTTAATTATTTTGATTGCTGGAATAAAGCATGTGGAAATGACCCAAACATAATCCGCAGTGGCCTCCTAATTTCCTTCTTTGGAGTTGGGGGAGGGGTAGACATGGGGAAGGGGCTTTGGGGTGATGGGCTTGCCTTCCATTCCTGCCCTTTCCCTCCCCACTATTCTCTTCTAGATCCCTCCATAACCCCACTCCCCTTTCTCTCACCCTTCTTATACCGCAAACCTTTCTACTTCCTCTTTCATTTTCTATTCTTGCAATTTCCTTGCACCTTTTCCAAATCCTCTTCTCCCCTGCAATACCATACAGGCAATCCACGTGCACAACACACACACACACTCTTCACATCTGGGGTTGTCCAAACCTCATACCCACTCCCCTTCAAGCCCATCCACTCTCCACCCCCTGGATGCCCTGCACTTGGTGGCGGTGGGATGCTCATGGATACTGGGAGGGTGAGGGGAGTGGAACCCGTGAGGAGGACCTGGGGGCCTCTCCTTGAACTGACATGAAGGGTCATCTGGCCTCTGCTCCCTTCTCACCCACGCTGACCTCCTGCCGAAGGAGCAACGCAACAGGAGAGGGGTCTGCTGAGCCTGGCGAGGGTCTGGGAGGGACCAGGAGGAAGGCGTGCTCCCTGCTCGCTGTCCTGGCCCTGGGGGAGTGAGGGAGACAGACACCTGGGAGAGCTGTGGGGAAGGCACTCGCACCGTGCTCTTGGGAAGGAAGGAGACCTGGCCCTGCTCACCACGGACTGGGTGCCTCGACCTCCTGAATCCCCAGAACACAACCCCCCTGGGCTGGGGTGGTCTGGGGAACCATCGTGCCCCCGCCTCCCG CCTACTCCTTTTTAAGCTT 3UTR-015Plod1; TTGGCCAGGCCTGACCCTCTTGGACCTTTCTTCTTTGCCGACA 246 procollagen-ACCACTGCCCAGCAGCCTCTGGGACCTCGGGGTCCCAGGGAAC lysine, 2-CCAGTCCAGCCTCCTGGCTGTTGACTTCCCATTGCTCTTGGAG oxoglutarateCCACCAATCAAAGAGATTCAAAGAGATTCCTGCAGGCCAGAGG 5-CGGAACACACCTTTATGGCTGGGGCTCTCCGTGGTGTTCTGGA dioxygenaseCCCAGCCCCTGGAGACACCATTCACTTTTACTGCTTTGTAGTG 1ACTCGTGCTCTCCAACCTGTCTTCCTGAAAAACCAAGGCCCCCTTCCCCCACCTCTTCCATGGGGTGAGACTTGAGCAGAACAGGGGCTTCCCCAAGTTGCCCAGAAAGACTGTCTGGGTGAGAAGCCATGGCCAGAGCTTCTCCCAGGCACAGGTGTTGCACCAGGGACTTCTGCTTCAAGTTTTGGGGTAAAGACACCTGGATCAGACTCCAAGGGCTGCCCTGAGTCTGGGACTTCTGCCTCCATGGCTGGTCATGAGAGCAAACCGTAGTCCCCTGGAGACAGCGACTCCAGAGAACCTCTTGGGAGACAGAAGAGGCATCTGTGCACAGCTCGATCTTCTACTTGCCTGTGGGGAGGGGAGTGACAGGTCCACACACCACACTGGGTCACCCTGTCCTGGATGCCTCTGAAGAGAGGGACAGACCGTCAGAAACTGGAGAGTTTCTATTAAAGGTCATTTAAACCA 3UTR-016 Nucb1;TCCTCCGGGACCCCAGCCCTCAGGATTCCTGATGCTCCAAGGC 247 nucleo-GACTGATGGGCGCTGGATGAAGTGGCACAGTCAGCTTCCCTGG bindin 1GGGCTGGTGTCATGTTGGGCTCCTGGGGCGGGGGCACGGCCTGGCATTTCACGCATTGCTGCCACCCCAGGTCCACCTGTCTCCACTTTCACAGCCTCCAAGTCTGTGGCTCTTCCCTTCTGTCCTCCGAGGGGCTTGCCTTCTCTCGTGTCCAGTGAGGTGCTCAGTGATCGGCTTAACTTAGAGAAGCCCGCCCCCTCCCCTTCTCCGTCTGTCCCAAGAGGGTCTGCTCTGAGCCTGCGTTCCTAGGTGGCTCGGCCTCAGCTGCCTGGGTTGTGGCCGCCCTAGCATCCTGTATGCCACCAGCTACTGGAATCCCCGCTGCTGCTCCGGGCCAAGCTTCTGGTTGATTAATGAGGGCATGGGGTGGTCCCTCAAGACCTTCCCCTACCTTTTGTGGAACCAGTGATGCCTCAAAGACAGTGTCCCCTCCACAGCTGGGTGCCAGGGGCAGGGGATCCTCAGTATAGCCGGTGAACCCTGATACCAGGAGCCTGGGCCTCCCTGAACCCCTGGCTTCCAGCCATCTCATCGCCAGCCTCCTCCTGGACCTCTTGGCCCCCAGCCCCTTCCCCACACAGCCCCAGAAGGGTCCCAGAGCTGACCCCACTCCAGGACCTAGGCCCAGCCCCTCAGCCTCATCTGGAGCCCCTGAAGACCAGTCCCACCCACCTTTCTGGCCTCATCTGACACTGCTCCGCATCCTGCTGTGTGTCCTGTTCCATGTTCCGGTTCCATCCAAATACACTTTCTGGAACAAA 3UTR-017 α-globinGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCC 248CCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCT TTGAATAAAGTCTGAGTGGGCGGC3UTR-018 TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTT 267GGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC 3UTR-019TGATAATAGTCCATAAAGTAGGAAACACTACAGCTGGAGCCTC 773GGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCCGCATTATTACTCACGGTACGAGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC 3UTR-020TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTT 774GGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCCGCATTATTACTCACGGTACGAGTGGTCTTTGAATAAAGTC TGAGTGGGCGGC

In certain embodiments, the 3′ UTR useful for the polynucleotidescomprises SEQ ID NO: 267.

In certain embodiments, the 5′UTR and/or 3′UTR sequence of thedisclosure comprises a nucleotide sequence at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, at leastabout 99%, or about 100% identical to a sequence selected from the groupconsisting of 5′UTR sequences comprising any of SEQ ID NOs: 215-231and/or 3′UTR sequences comprises any of SEQ ID NOs: 232-248, and anycombination thereof. The polynucleotides of the disclosure can comprisecombinations of features. For example, the ORF can be flanked by a 5′UTRthat comprises a strong Kozak translational initiation signal and/or a3′UTR comprising an oligo(dT) sequence for templated addition of apoly-A tail. A 5′UTR can comprise a first polynucleotide fragment and asecond polynucleotide fragment from the same and/or different UTRs (see,e.g., US2010/0293625, herein incorporated by reference in its entirety).

It is also within the scope of the present disclosure to have patternedUTRs. As used herein “patterned UTRs” include a repeating or alternatingpattern, such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereofrepeated once, twice, or more than 3 times. In these patterns, eachletter, A, B, or C represent a different UTR nucleic acid sequence.

Other non-UTR sequences can be used as regions or subregions within thepolynucleotides of the disclosure. For example, introns or portions ofintron sequences can be incorporated into the polynucleotides of thedisclosure. Incorporation of intronic sequences can increase proteinproduction as well as polynucleotide expression levels. In someembodiments, the polynucleotide of the disclosure comprises an internalribosome entry site (IRES) instead of or in addition to a UTR (see,e.g., Yakubov et al., Biochem. Biophys. Res. Commun. 2010394(1):189-193, the contents of which are incorporated herein byreference in their entirety). In some embodiments, the polynucleotide ofthe disclosure comprises 5′ and/or 3′ sequence associated with the 5′and/or 3′ ends of rubella virus (RV) genomic RNA, respectively, ordeletion derivatives thereof, including the 5′ proximal open readingframe of RV RNA encoding nonstructural proteins (e.g., see Pogue et al.,J. Virol. 67(12):7106-7117, the contents of which are incorporatedherein by reference in their entirety). Viral capsid sequences can alsobe used as a translational enhancer, e.g., the 5′ portion of a capsidsequence, (e.g., semliki forest virus and sindbis virus capsid RNAs asdescribed in Sjöberg et al., Biotechnology (NY) 1994 12(11):1127-1131,and Frolov and Schlesinger J. Virol. 1996 70(2):1182-1190, the contentsof each of which are incorporated herein by reference in theirentirety). In some embodiments, the polynucleotide comprises an IRESinstead of a 5′UTR sequence. In some embodiments, the polynucleotidecomprises an ORF and a viral capsid sequence. In some embodiments, thepolynucleotide comprises a synthetic 5′UTR in combination with anon-synthetic 3′UTR.

In some embodiments, the UTR can also include at least one translationenhancer polynucleotide, translation enhancer element, or translationalenhancer elements (collectively, “TEE,” which refers to nucleic acidsequences that increase the amount of polypeptide or protein producedfrom a polynucleotide. As a non-limiting example, the TEE can includethose described in US2009/0226470, incorporated herein by reference inits entirety, and others known in the art. As a non-limiting example,the TEE can be located between the transcription promoter and the startcodon. In some embodiments, the 5′UTR comprises a TEE.

In one aspect, a TEE is a conserved element in a UTR that can promotetranslational activity of a nucleic acid such as, but not limited to,cap-dependent or cap-independent translation. The conservation of thesesequences has been shown across 14 species including humans. See, e.g.,Panek et al., “An evolutionary conserved pattern of 18S rRNA sequencecomplementarity to mRNA 5′UTRs and its implications for eukaryotic genetranslation regulation,” Nucleic Acids Research 2013,doi:10.1093/nar/gkt548, incorporated herein by reference in itsentirety.

In one non-limiting example, the TEE comprises the TEE sequence in the5′-leader of the Gtx homeodomain protein. See Chappell et al., PNAS 2004101:9590-9594, incorporated herein by reference in its entirety.

In another non-limiting example, the TEE comprises a TEE having one ormore of the sequences of SEQ ID NOs: 1-35 in US2009/0226470,US2013/0177581, and WO2009/075886; SEQ ID NOs: 1-5 and 7-645 inWO2012/009644; and SEQ ID NO: 1 WO1999/024595, U.S. Pat. No. 6,310,197,and U.S. Pat. No. 6,849,405; the contents of each of which areincorporated herein by reference in their entirety.

In some embodiments, the TEE is an internal ribosome entry site (IRES),HCV-IRES, or an IRES element such as, but not limited to, thosedescribed in: U.S. Pat. No. 7,468,275, US2007/0048776, US2011/0124100,WO2007/025008, and WO2001/055369; the contents of each of which reincorporated herein by reference in their entirety. The IRES elementscan include, but are not limited to, the Gtx sequences (e.g., Gtx9-nt,Gtx8-nt, Gtx7-nt) as described by Chappell et al., PNAS 2004101:9590-9594, Zhou et al., PNAS 2005 102:6273-6278, US2007/0048776,US2011/0124100, and WO2007/025008; the contents of each of which areincorporated herein by reference in their entirety.

“Translational enhancer polynucleotide” or “translation enhancerpolynucleotide sequence” refer to a polynucleotide that includes one ormore of the TEE provided herein and/or known in the art (see. e.g., U.S.Pat. No. 6,310,197, U.S. Pat. No. 6,849,405, U.S. Pat. No. 7,456,273,U.S. Pat. No. 7,183,395, US2009/0226470, US2007/0048776, US2011/0124100,US2009/0093049, US2013/0177581, WO2009/075886, WO2007/025008,WO2012/009644, WO2001/055371, WO1999/024595, EP2610341A1, andEP2610340A1; the contents of each of which are incorporated herein byreference in their entirety), or their variants, homologs, or functionalderivatives. In some embodiments, the polynucleotide of the disclosurecomprises one or multiple copies of a TEE. The TEE in a translationalenhancer polynucleotide can be organized in one or more sequencesegments. A sequence segment can harbor one or more of the TEEs providedherein, with each TEE being present in one or more copies. When multiplesequence segments are present in a translational enhancerpolynucleotide, they can be homogenous or heterogeneous. Thus, themultiple sequence segments in a translational enhancer polynucleotidecan harbor identical or different types of the TEE provided herein,identical or different number of copies of each of the TEE, and/oridentical or different organization of the TEE within each sequencesegment. In one embodiment, the polynucleotide of the disclosurecomprises a translational enhancer polynucleotide sequence.

In some embodiments, a 5′UTR and/or 3′UTR of a polynucleotide of thedisclosure comprises at least one TEE or portion thereof that isdisclosed in: WO1999/024595, WO2012/009644, WO2009/075886,WO2007/025008, WO1999/024595, WO2001/055371, EP2610341A1, EP2610340A1,U.S. Pat. No. 6,310,197, U.S. Pat. No. 6,849,405, U.S. Pat. No.7,456,273, U.S. Pat. No. 7,183,395, US2009/0226470, US2011/0124100,US2007/0048776, US2009/0093049, or US2013/0177581, the contents of eachare incorporated herein by reference in their entirety.

In some embodiments, a 5′UTR and/or 3′UTR of a polynucleotide of thedisclosure comprises a TEE that is at least 5%, at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% identical to a TEE disclosed in: US2009/0226470,US2007/0048776, US2013/0177581, US2011/0124100, WO1999/024595,WO2012/009644, WO2009/075886, WO2007/025008, EP2610341A1, EP2610340A1,U.S. Pat. No. 6,310,197, U.S. Pat. No. 6,849,405, U.S. Pat. No.7,456,273, U.S. Pat. No. 7,183,395, Chappell et al., PNAS 2004101:9590-9594, Zhou et al., PNAS 2005 102:6273-6278, and SupplementalTable 1 and in Supplemental Table 2 of Wellensiek et al., “Genome-wideprofiling of human cap-independent translation-enhancing elements,”Nature Methods 2013, DOI:10.1038/NMETH.2522; the contents of each ofwhich are incorporated herein by reference in their entirety.

In some embodiments, a 5′UTR and/or 3′UTR of a polynucleotide of thedisclosure comprises a TEE which is selected from a 5-30 nucleotidefragment, a 5-25 nucleotide fragment, a 5-20 nucleotide fragment, a 5-15nucleotide fragment, or a 5-10 nucleotide fragment (including a fragmentof 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) of a TEE sequencedisclosed in: US2009/0226470, US2007/0048776, US2013/0177581,US2011/0124100, WO1999/024595, WO2012/009644, WO2009/075886,WO2007/025008, EP2610341A1, EP2610340A1, U.S. Pat. No. 6,310,197, U.S.Pat. No. 6,849,405, U.S. Pat. No. 7,456,273, U.S. Pat. No. 7,183,395,Chappell et al., PNAS 2004 101:9590-9594, Zhou et al., PNAS 2005102:6273-6278, and Supplemental Table 1 and in Supplemental Table 2 ofWellensiek et al., “Genome-wide profiling of human cap-independenttranslation-enhancing elements,” Nature Methods 2013,DOI:10.1038/NMETH.2522.

In some embodiments, a 5′UTR and/or 3′UTR of a polynucleotide of thedisclosure comprises a TEE which is a transcription regulatory elementdescribed in any of U.S. Pat. No. 7,456,273, U.S. Pat. No. 7,183,395,US2009/0093049, and WO2001/055371, the contents of each of which areincorporated herein by reference in their entirety. The transcriptionregulatory elements can be identified by methods known in the art, suchas, but not limited to, the methods described in U.S. Pat. No.7,456,273, U.S. Pat. No. 7,183,395, US2009/0093049, and WO2001/055371.

In some embodiments, a 5′UTR and/or 3′UTR comprising at least one TEEdescribed herein can be incorporated in a monocistronic sequence suchas, but not limited to, a vector system or a nucleic acid vector. Asnon-limiting examples, the vector systems and nucleic acid vectors caninclude those described in U.S. Pat. No. 7,456,273, U.S. Pat. No.7,183,395, US2007/0048776, US2009/0093049, US2011/0124100,WO2007/025008, and WO2001/055371.

In some embodiments, a 5′UTR and/or 3′UTR of a polynucleotide of thedisclosure comprises a TEE or portion thereof described herein. In someembodiments, the TEEs in the 3′UTR can be the same and/or different fromthe TEE located in the 5′UTR.

In some embodiments, a 5′UTR and/or 3′UTR of a polynucleotide of thedisclosure can include at least 1, at least 2, at least 3, at least 4,at least 5, at least 6, at least 7, at least 8, at least 9, at least 10,at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18 at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 30, atleast 35, at least 40, at least 45, at least 50, at least 55 or morethan 60 TEE sequences. In one embodiment, the 5′UTR of a polynucleotideof the disclosure can include 1-60, 1-55, 1-50, 1-45, 1-40, 1-35, 1-30,1-25, 1-20, 1-15, 1-10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 TEE sequences. TheTEE sequences in the 5′UTR of the polynucleotide of the disclosure canbe the same or different TEE sequences. A combination of different TEEsequences in the 5′UTR of the polynucleotide of the disclosure caninclude combinations in which more than one copy of any of the differentTEE sequences are incorporated. The TEE sequences can be in a patternsuch as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeatedone, two, three, or more than three times. In these patterns, eachletter, A, B, or C represent a different TEE nucleotide sequence.

In some embodiments, the TEE can be identified by the methods describedin US2007/0048776, US2011/0124100, WO2007/025008, WO2012/009644, thecontents of each of which are incorporated herein by reference in theirentirety.

In some embodiments, the 5′UTR and/or 3′UTR comprises a spacer toseparate two TEE sequences. As a non-limiting example, the spacer can bea 15 nucleotide spacer and/or other spacers known in the art. As anothernon-limiting example, the 5′UTR and/or 3′UTR comprises a TEEsequence-spacer module repeated at least once, at least twice, at least3 times, at least 4 times, at least 5 times, at least 6 times, at least7 times, at least 8 times, at least 9 times, at least 10 times, or morethan 10 times in the 5′UTR and/or 3′UTR, respectively. In someembodiments, the 5′UTR and/or 3′UTR comprises a TEE sequence-spacermodule repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

In some embodiments, the spacer separating two TEE sequences can includeother sequences known in the art that can regulate the translation ofthe polynucleotide of the disclosure, e.g., miR sequences describedherein (e.g., miR binding sites and miR seeds). As a non-limitingexample, each spacer used to separate two TEE sequences can include adifferent miR sequence or component of a miR sequence (e.g., miR seedsequence).

In some embodiments, a polynucleotide of the disclosure comprises a miRand/or TEE sequence. In some embodiments, the incorporation of a miRsequence and/or a TEE sequence into a polynucleotide of the disclosurecan change the shape of the stem loop region, which can increase and/ordecrease translation. See e.g., Kedde et al., Nature Cell Biology 201012(10):1014-20, herein incorporated by reference in its entirety).

Sensor Sequences and MicroRNA (miRNA) Binding Sites

Polynucleotides of the disclosure can include regulatory elements, forexample, microRNA (miRNA) binding sites, transcription factor bindingsites, structured mRNA sequences and/or motifs, artificial binding sitesengineered to act as pseudo-receptors for endogenous nucleic acidbinding molecules, and combinations thereof. In some embodiments,polynucleotides including such regulatory elements are referred to asincluding “sensor sequences”. Non-limiting examples of sensor sequencesare described in U.S. Publication 2014/0200261, the contents of whichare incorporated herein by reference in their entirety.

In some embodiments, a polynucleotide (e.g., a ribonucleic acid (RNA),e.g., a messenger RNA (mRNA)) of the disclosure comprises an openreading frame (ORF) encoding a polypeptide of interest and furthercomprises one or more miRNA binding site(s). Inclusion or incorporationof miRNA binding site(s) provides for regulation of polynucleotides ofthe disclosure, and in turn, of the polypeptides encoded therefrom,based on tissue-specific and/or cell-type specific expression ofnaturally-occurring miRNAs.

A miRNA, e.g., a natural-occurring miRNA, is a 19-25 nucleotide longnoncoding RNA that binds to a polynucleotide and down-regulates geneexpression either by reducing stability or by inhibiting translation ofthe polynucleotide. A miRNA sequence comprises a “seed” region, i.e., asequence in the region of positions 2-8 of the mature miRNA. A miRNAseed can comprise positions 2-8 or 2-7 of the mature miRNA. In someembodiments, a miRNA seed can comprise 7 nucleotides (e.g., nucleotides2-8 of the mature miRNA), wherein the seed-complementary site in thecorresponding miRNA binding site is flanked by an adenosine (A) opposedto miRNA position 1. In some embodiments, a miRNA seed can comprise 6nucleotides (e.g., nucleotides 2-7 of the mature miRNA), wherein theseed-complementary site in the corresponding miRNA binding site isflanked by an adenosine (A) opposed to miRNA position 1. See, forexample, Grimson A, Farh K K, Johnston W K, Garrett-Engele P, Lim L P,Bartel D P; Mol Cell. 2007 Jul. 6; 27(1):91-105. miRNA profiling of thetarget cells or tissues can be conducted to determine the presence orabsence of miRNA in the cells or tissues. In some embodiments, apolynucleotide (e.g., a ribonucleic acid (RNA), e.g., a messenger RNA(mRNA)) of the disclosure comprises one or more microRNA binding sites,microRNA target sequences, microRNA complementary sequences, or microRNAseed complementary sequences. Such sequences can correspond to, e.g.,have complementarity to, any known microRNA such as those taught in USPublication US2005/0261218 and US Publication US2005/0059005, thecontents of each of which are incorporated herein by reference in theirentirety.

As used herein, the term “microRNA (miRNA or miR) binding site” refersto a sequence within a polynucleotide, e.g., within a DNA or within anRNA transcript, including in the 5′UTR and/or 3′UTR, that has sufficientcomplementarity to all or a region of a miRNA to interact with,associate with or bind to the miRNA. In some embodiments, apolynucleotide of the disclosure comprising an ORF encoding apolypeptide of interest and further comprises one or more miRNA bindingsite(s). In exemplary embodiments, a 5′UTR and/or 3′UTR of thepolynucleotide (e.g., a ribonucleic acid (RNA), e.g., a messenger RNA(mRNA)) comprises the one or more miRNA binding site(s).

A miRNA binding site having sufficient complementarity to a miRNA refersto a degree of complementarity sufficient to facilitate miRNA-mediatedregulation of a polynucleotide, e.g., miRNA-mediated translationalrepression or degradation of the polynucleotide. In exemplary aspects ofthe disclosure, a miRNA binding site having sufficient complementarityto the miRNA refers to a degree of complementarity sufficient tofacilitate miRNA-mediated degradation of the polynucleotide, e.g.,miRNA-guided RNA-induced silencing complex (RISC)-mediated cleavage ofmRNA. The miRNA binding site can have complementarity to, for example, a19-25 nucleotide miRNA sequence, to a 19-23 nucleotide miRNA sequence,or to a 22 nucleotide miRNA sequence. A miRNA binding site can becomplementary to only a portion of a miRNA, e.g., to a portion less than1, 2, 3, or 4 nucleotides of the full length of a naturally-occurringmiRNA sequence. Full or complete complementarity (e.g., fullcomplementarity or complete complementarity over all or a significantportion of the length of a naturally-occurring miRNA) is preferred whenthe desired regulation is mRNA degradation.

In some embodiments, a miRNA binding site includes a sequence that hascomplementarity (e.g., partial or complete complementarity) with anmiRNA seed sequence. In some embodiments, the miRNA binding siteincludes a sequence that has complete complementarity with a miRNA seedsequence. In some embodiments, a miRNA binding site includes a sequencethat has complementarity (e.g., partial or complete complementarity)with an miRNA sequence. In some embodiments, the miRNA binding siteincludes a sequence that has complete complementarity with a miRNAsequence. In some embodiments, a miRNA binding site has completecomplementarity with a miRNA sequence but for 1, 2, or 3 nucleotidesubstitutions, terminal additions, and/or truncations.

In some embodiments, the miRNA binding site is the same length as thecorresponding miRNA. In other embodiments, the miRNA binding site isone, two, three, four, five, six, seven, eight, nine, ten, eleven ortwelve nucleotide(s) shorter than the corresponding miRNA at the 5′terminus, the 3′ terminus, or both. In still other embodiments, themicroRNA binding site is two nucleotides shorter than the correspondingmicroRNA at the 5′ terminus, the 3′ terminus, or both. The miRNA bindingsites that are shorter than the corresponding miRNAs are still capableof degrading the mRNA incorporating one or more of the miRNA bindingsites or preventing the mRNA from translation.

In some embodiments, the miRNA binding site binds the correspondingmature miRNA that is part of an active RISC containing Dicer. In anotherembodiment, binding of the miRNA binding site to the corresponding miRNAin RISC degrades the mRNA containing the miRNA binding site or preventsthe mRNA from being translated. In some embodiments, the miRNA bindingsite has sufficient complementarity to miRNA so that a RISC complexcomprising the miRNA cleaves the polynucleotide comprising the miRNAbinding site. In other embodiments, the miRNA binding site has imperfectcomplementarity so that a RISC complex comprising the miRNA inducesinstability in the polynucleotide comprising the miRNA binding site. Inanother embodiment, the miRNA binding site has imperfect complementarityso that a RISC complex comprising the miRNA represses transcription ofthe polynucleotide comprising the miRNA binding site.

In some embodiments, the miRNA binding site has one, two, three, four,five, six, seven, eight, nine, ten, eleven or twelve mismatch(es) fromthe corresponding miRNA.

In some embodiments, the miRNA binding site has at least about ten, atleast about eleven, at least about twelve, at least about thirteen, atleast about fourteen, at least about fifteen, at least about sixteen, atleast about seventeen, at least about eighteen, at least about nineteen,at least about twenty, or at least about twenty-one contiguousnucleotides complementary to at least about ten, at least about eleven,at least about twelve, at least about thirteen, at least about fourteen,at least about fifteen, at least about sixteen, at least aboutseventeen, at least about eighteen, at least about nineteen, at leastabout twenty, or at least about twenty-one, respectively, contiguousnucleotides of the corresponding miRNA.

By engineering one or more miRNA binding sites into a polynucleotide ofthe disclosure, the polynucleotide can be targeted for degradation orreduced translation, provided the miRNA in question is available. Thiscan reduce off-target effects upon delivery of the polynucleotide. Forexample, if a polynucleotide of the disclosure is not intended to bedelivered to a tissue or cell but ends up is said tissue or cell, then amiRNA abundant in the tissue or cell can inhibit the expression of thegene of interest if one or multiple binding sites of the miRNA areengineered into the 5′UTR and/or 3′UTR of the polynucleotide.

Conversely, miRNA binding sites can be removed from polynucleotidesequences in which they naturally occur in order to increase proteinexpression in specific tissues. For example, a binding site for aspecific miRNA can be removed from a polynucleotide to improve proteinexpression in tissues or cells containing the miRNA.

In one embodiment, a polynucleotide of the disclosure can include atleast one miRNA-binding site in the 5′UTR and/or 3′UTR in order toregulate cytotoxic or cytoprotective mRNA therapeutics to specific cellssuch as, but not limited to, normal and/or cancerous cells. In anotherembodiment, a polynucleotide of the disclosure can include two, three,four, five, six, seven, eight, nine, ten, or more miRNA-binding sites inthe 5′-UTR and/or 3′-UTR in order to regulate cytotoxic orcytoprotective mRNA therapeutics to specific cells such as, but notlimited to, normal and/or cancerous cells.

Regulation of expression in multiple tissues can be accomplished throughintroduction or removal of one or more miRNA binding sites, e.g., one ormore distinct miRNA binding sites. The decision whether to remove orinsert a miRNA binding site can be made based on miRNA expressionpatterns and/or their profilings in tissues and/or cells in developmentand/or disease. Identification of miRNAs, miRNA binding sites, and theirexpression patterns and role in biology have been reported (e.g.,Bonauer et al., Curr Drug Targets 2010 11:943-949; Anand and ChereshCurr Opin Hematol 2011 18:171-176; Contreras and Rao Leukemia 201226:404-413 (2011 Dec. 20. doi: 10.1038/leu.2011.356); Bartel Cell 2009136:215-233; Landgraf et al, Cell, 2007 129:1401-1414; Gentner andNaldini, Tissue Antigens. 2012 80:393-403 and all references therein;each of which is incorporated herein by reference in its entirety).

miRNAs and miRNA binding sites can correspond to any known sequence,including non-limiting examples described in U.S. Publication Nos.2014/0200261, 2005/0261218, and 2005/0059005, each of which areincorporated herein by reference in their entirety.

Examples of tissues where miRNA are known to regulate mRNA, and therebyprotein expression, include, but are not limited to, liver (miR-122),muscle (miR-133, miR-206, miR-208), endothelial cells (miR-17-92,miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR-16, miR-21,miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart(miR-1d, miR-149), kidney (miR-192, miR-194, miR-204), and lungepithelial cells (let-7, miR-133, miR-126).

Specifically, miRNAs are known to be differentially expressed in immunecells (also called hematopoietic cells), such as antigen presentingcells (APCs) (e.g., dendritic cells and macrophages), macrophages,monocytes, B lymphocytes, T lymphocytes, granulocytes, natural killercells, etc. Immune cell specific miRNAs are involved in immunogenicity,autoimmunity, the immune-response to infection, inflammation, as well asunwanted immune response after gene therapy and tissue/organtransplantation. Immune cells specific miRNAs also regulate many aspectsof development, proliferation, differentiation and apoptosis ofhematopoietic cells (immune cells). For example, miR-142 and miR-146 areexclusively expressed in immune cells, particularly abundant in myeloiddendritic cells. It has been demonstrated that the immune response to apolynucleotide can be shut-off by adding miR-142 binding sites to the3′-UTR of the polynucleotide, enabling more stable gene transfer intissues and cells. miR-142 efficiently degrades exogenouspolynucleotides in antigen presenting cells and suppresses cytotoxicelimination of transduced cells (e.g., Annoni A et al., blood, 2009,114, 5152-5161; Brown B D, et al., Nat med. 2006, 12(5), 585-591; BrownB D, et al., blood, 2007, 110(13): 4144-4152, each of which isincorporated herein by reference in its entirety).

An antigen-mediated immune response can refer to an immune responsetriggered by foreign antigens, which, when entering an organism, areprocessed by the antigen presenting cells and displayed on the surfaceof the antigen presenting cells. T cells can recognize the presentedantigen and induce a cytotoxic elimination of cells that express theantigen.

Introducing a miR-142 binding site into the 5′UTR and/or 3′UTR of apolynucleotide of the disclosure can selectively repress gene expressionin antigen presenting cells through miR-142 mediated degradation,limiting antigen presentation in antigen presenting cells (e.g.,dendritic cells) and thereby preventing antigen-mediated immune responseafter the delivery of the polynucleotide. The polynucleotide is thenstably expressed in target tissues or cells without triggering cytotoxicelimination.

In one embodiment, binding sites for miRNAs that are known to beexpressed in immune cells, in particular, antigen presenting cells, canbe engineered into a polynucleotide of the disclosure to suppress theexpression of the polynucleotide in antigen presenting cells throughmiRNA mediated RNA degradation, subduing the antigen-mediated immuneresponse. Expression of the polynucleotide is maintained in non-immunecells where the immune cell specific miRNAs are not expressed. Forexample, in some embodiments, to prevent an immunogenic reaction againsta liver specific protein, any miR-122 binding site can be removed and amiR-142 (and/or mirR-146) binding site can be engineered into the 5′UTRand/or 3′UTR of a polynucleotide of the disclosure.

To further drive the selective degradation and suppression in APCs andmacrophage, a polynucleotide of the disclosure can include a furthernegative regulatory element in the 5′UTR and/or 3′UTR, either alone orin combination with miR-142 and/or miR-146 binding sites. As anon-limiting example, the further negative regulatory element is aConstitutive Decay Element (CDE).

Immune cell specific miRNAs include, but are not limited to,hsa-let-7a-2-3p, hsa-let-7a-3p, hsa-7a-5p, hsa-let-7c, hsa-let-7e-3p,hsa-let-7e-5p, hsa-let-7g-3p, hsa-let-7g-5p, hsa-let-7i-3p,hsa-let-7i-5p, miR-110a-3p, miR-110a-5p, miR-1184, hsa-let-7f-1-3p,hsa-let-7f-2-5p, hsa-let-7f-5p, miR-125b-1-3p, miR-125b-2-3p,miR-125b-5p, miR-1279, miR-130a-3p, miR-130a-5p, miR-132-3p, miR-132-5p,miR-142-3p, miR-142-5p, miR-143-3p, miR-143-5p, miR-146a-3p,miR-146a-5p, miR-146b-3p, miR-146b-5p, miR-147a, miR-147b, miR-148a-5p,miR-148a-3p, miR-150-3p, miR-150-5p, miR-151b, miR-155-3p, miR-155-5p,miR-15a-3p, miR-15a-5p, miR-15b-5p, miR-15b-3p, miR-16-1-3p,miR-16-2-3p, miR-16-5p, miR-17-5p, miR-181a-3p, miR-181a-5p,miR-181a-2-3p, miR-182-3p, miR-182-5p, miR-197-3p, miR-197-5p,miR-21-5p, miR-21-3p, miR-214-3p, miR-214-5p, miR-223-3p, miR-223-5p,miR-221-3p, miR-221-5p, miR-23b-3p, miR-23b-5p, miR-24-1-5p,miR-24-2-5p, miR-24-3p, miR-26a-1-3p, miR-26a-2-3p, miR-26a-5p,miR-26b-3p, miR-26b-5p, miR-27a-3p, miR-27a-5p, miR-27b-3p, miR-27b-5p,miR-28-3p, miR-28-5p, miR-2909, miR-29a-3p, miR-29a-5p, miR-29b-1-5p,miR-29b-2-5p, miR-29c-3p, miR-29c-5p, miR-30e-3p, miR-30e-5p,miR-331-5p, miR-339-3p, miR-339-5p, miR-345-3p, miR-345-5p, miR-346,miR-34a-3p, miR-34a-5p, miR-363-3p, miR-363-5p, miR-372, miR-377-3p,miR-377-5p, miR-493-3p, miR-493-5p, miR-542, miR-548b-5p, miR548c-5p,miR-548i, miR-548j, miR-548n, miR-574-3p, miR-598, miR-718, miR-935,miR-99a-3p, miR-99a-5p, miR-99b-3p, and miR-99b-5p. Furthermore, novelmiRNAs can be identified in immune cell through micro-arrayhybridization and microtome analysis (e.g., Jima D D et al, Blood, 2010,116:e118-e127; Vaz C et al., BMC Genomics, 2010, 11,288, the content ofeach of which is incorporated herein by reference in its entirety.)

miRNAs that are known to be expressed in the liver include, but are notlimited to, miR-107, miR-122-3p, miR-122-5p, miR-1228-3p, miR-1228-5p,miR-1249, miR-129-5p, miR-1303, miR-151a-3p, miR-151a-5p, miR-152,miR-194-3p, miR-194-5p, miR-199a-3p, miR-199a-5p, miR-199b-3p,miR-199b-5p, miR-296-5p, miR-557, miR-581, miR-939-3p, and miR-939-5p.MiRNA binding sites from any liver specific miRNA can be introduced toor removed from a polynucleotide of the disclosure to regulateexpression of the polynucleotide in the liver. Liver specific miRNAbinding sites can be engineered alone or further in combination withimmune cell (e.g., APC) miRNA binding sites in a polynucleotide of thedisclosure.

miRNAs that are known to be expressed in the lung include, but are notlimited to, let-7a-2-3p, let-7a-3p, let-7a-5p, miR-126-3p, miR-126-5p,miR-127-3p, miR-127-5p, miR-130a-3p, miR-130a-5p, miR-130b-3p,miR-130b-5p, miR-133a, miR-133b, miR-134, miR-18a-3p, miR-18a-5p,miR-18b-3p, miR-18b-5p, miR-24-1-5p, miR-24-2-5p, miR-24-3p, miR-296-3p,miR-296-5p, miR-32-3p, miR-337-3p, miR-337-5p, miR-381-3p, andmiR-381-5p. miRNA binding sites from any lung specific miRNA can beintroduced to or removed from a polynucleotide of the disclosure toregulate expression of the polynucleotide in the lung. Lung specificmiRNA binding sites can be engineered alone or further in combinationwith immune cell (e.g., APC) miRNA binding sites in a polynucleotide ofthe disclosure.

miRNAs that are known to be expressed in the heart include, but are notlimited to, miR-1, miR-133a, miR-133b, miR-149-3p, miR-149-5p,miR-186-3p, miR-186-5p, miR-208a, miR-208b, miR-210, miR-296-3p,miR-320, miR-451a, miR-451b, miR-499a-3p, miR-499a-5p, miR-499b-3p,miR-499b-5p, miR-744-3p, miR-744-5p, miR-92b-3p, and miR-92b-5p. mMiRNAbinding sites from any heart specific microRNA can be introduced to orremoved from a polynucleotide of the disclosure to regulate expressionof the polynucleotide in the heart. Heart specific miRNA binding sitescan be engineered alone or further in combination with immune cell(e.g., APC) miRNA binding sites in a polynucleotide of the disclosure.

miRNAs that are known to be expressed in the nervous system include, butare not limited to, miR-124-5p, miR-125a-3p, miR-125a-5p, miR-125b-1-3p,miR-125b-2-3p, miR-125b-5p, miR-1271-3p, miR-1271-5p, miR-128,miR-132-5p, miR-135a-3p, miR-135a-5p, miR-135b-3p, miR-135b-5p, miR-137,miR-139-5p, miR-139-3p, miR-149-3p, miR-149-5p, miR-153, miR-181c-3p,miR-181c-5p, miR-183-3p, miR-183-5p, miR-190a, miR-190b, miR-212-3p,miR-212-5p, miR-219-1-3p, miR-219-2-3p, miR-23a-3p, miR-23a-5p,miR-30a-5p, miR-30b-3p, miR-30b-5p, miR-30c-1-3p, miR-30c-2-3p,miR-30c-5p, miR-30d-3p, miR-30d-5p, miR-329, miR-342-3p, miR-3665,miR-3666, miR-380-3p, miR-380-5p, miR-383, miR-410, miR-425-3p,miR-425-5p, miR-454-3p, miR-454-5p, miR-483, miR-510, miR-516a-3p,miR-548b-5p, miR-548c-5p, miR-571, miR-7-1-3p, miR-7-2-3p, miR-7-5p,miR-802, miR-922, miR-9-3p, and miR-9-5p. miRNAs enriched in the nervoussystem further include those specifically expressed in neurons,including, but not limited to, miR-132-3p, miR-132-3p, miR-148b-3p,miR-148b-5p, miR-151a-3p, miR-151a-5p, miR-212-3p, miR-212-5p, miR-320b,miR-320e, miR-323a-3p, miR-323a-5p, miR-324-5p, miR-325, miR-326,miR-328, miR-922 and those specifically expressed in glial cells,including, but not limited to, miR-1250, miR-219-1-3p, miR-219-2-3p,miR-219-5p, miR-23a-3p, miR-23a-5p, miR-3065-3p, miR-3065-5p,miR-30e-3p, miR-30e-5p, miR-32-5p, miR-338-5p, and miR-657. miRNAbinding sites from any CNS specific miRNA can be introduced to orremoved from a polynucleotide of the disclosure to regulate expressionof the polynucleotide in the nervous system. Nervous system specificmiRNA binding sites can be engineered alone or further in combinationwith immune cell (e.g., APC) miRNA binding sites in a polynucleotide ofthe disclosure.

miRNAs that are known to be expressed in the pancreas include, but arenot limited to, miR-105-3p, miR-105-5p, miR-184, miR-195-3p, miR-195-5p,miR-196a-3p, miR-196a-5p, miR-214-3p, miR-214-5p, miR-216a-3p,miR-216a-5p, miR-30a-3p, miR-33a-3p, miR-33a-5p, miR-375, miR-7-1-3p,miR-7-2-3p, miR-493-3p, miR-493-5p, and miR-944. MiRNA binding sitesfrom any pancreas specific miRNA can be introduced to or removed from apolynucleotide of the disclosure to regulate expression of thepolynucleotide in the pancreas. Pancreas specific miRNA binding sitescan be engineered alone or further in combination with immune cell (e.g.APC) miRNA binding sites in a polynucleotide of the disclosure.

miRNAs that are known to be expressed in the kidney include, but are notlimited to, miR-122-3p, miR-145-5p, miR-17-5p, miR-192-3p, miR-192-5p,miR-194-3p, miR-194-5p, miR-20a-3p, miR-20a-5p, miR-204-3p, miR-204-5p,miR-210, miR-216a-3p, miR-216a-5p, miR-296-3p, miR-30a-3p, miR-30a-5p,miR-30b-3p, miR-30b-5p, miR-30c-1-3p, miR-30c-2-3p, miR30c-5p,miR-324-3p, miR-335-3p, miR-335-5p, miR-363-3p, miR-363-5p, and miR-562.miRNA binding sites from any kidney specific miRNA can be introduced toor removed from a polynucleotide of the disclosure to regulateexpression of the polynucleotide in the kidney. Kidney specific miRNAbinding sites can be engineered alone or further in combination withimmune cell (e.g., APC) miRNA binding sites in a polynucleotide of thedisclosure.

miRNAs that are known to be expressed in the muscle include, but are notlimited to, let-7g-3p, let-7g-5p, miR-1, miR-1286, miR-133a, miR-133b,miR-140-3p, miR-143-3p, miR-143-5p, miR-145-3p, miR-145-5p, miR-188-3p,miR-188-5p, miR-206, miR-208a, miR-208b, miR-25-3p, and miR-25-5p. MiRNAbinding sites from any muscle specific miRNA can be introduced to orremoved from a polynucleotide of the disclosure to regulate expressionof the polynucleotide in the muscle. Muscle specific miRNA binding sitescan be engineered alone or further in combination with immune cell(e.g., APC) miRNA binding sites in a polynucleotide of the disclosure.

miRNAs are also differentially expressed in different types of cells,such as, but not limited to, endothelial cells, epithelial cells, andadipocytes.

miRNAs that are known to be expressed in endothelial cells include, butare not limited to, let-7b-3p, let-7b-5p, miR-100-3p, miR-100-5p,miR-101-3p, miR-101-5p, miR-126-3p, miR-126-5p, miR-1236-3p,miR-1236-5p, miR-130a-3p, miR-130a-5p, miR-17-5p, miR-17-3p, miR-18a-3p,miR-18a-5p, miR-19a-3p, miR-19a-5p, miR-19b-1-5p, miR-19b-2-5p,miR-19b-3p, miR-20a-3p, miR-20a-5p, miR-217, miR-210, miR-21-3p,miR-21-5p, miR-221-3p, miR-221-5p, miR-222-3p, miR-222-5p, miR-23a-3p,miR-23a-5p, miR-296-5p, miR-361-3p, miR-361-5p, miR-421, miR-424-3p,miR-424-5p, miR-513a-5p, miR-92a-1-5p, miR-92a-2-5p, miR-92a-3p,miR-92b-3p, and miR-92b-5p. Many novel miRNAs are discovered inendothelial cells from deep-sequencing analysis (e.g., Voellenkle C etal., RNA, 2012, 18, 472-484, herein incorporated by reference in itsentirety). miRNA binding sites from any endothelial cell specific miRNAcan be introduced to or removed from a polynucleotide of the disclosureto regulate expression of the polynucleotide in the endothelial cells.

miRNAs that are known to be expressed in epithelial cells include, butare not limited to, let-7b-3p, let-7b-5p, miR-1246, miR-200a-3p,miR-200a-5p, miR-200b-3p, miR-200b-5p, miR-200c-3p, miR-200c-5p,miR-338-3p, miR-429, miR-451a, miR-451b, miR-494, miR-802 and miR-34a,miR-34b-5p, miR-34c-5p, miR-449a, miR-449b-3p, miR-449b-5p specific inrespiratory ciliated epithelial cells, let-7 family, miR-133a, miR-133b,miR-126 specific in lung epithelial cells, miR-382-3p, miR-382-5pspecific in renal epithelial cells, and miR-762 specific in cornealepithelial cells. miRNA binding sites from any epithelial cell specificmiRNA can be introduced to or removed from a polynucleotide of thedisclosure to regulate expression of the polynucleotide in theepithelial cells.

In addition, a large group of miRNAs are enriched in embryonic stemcells, controlling stem cell self-renewal as well as the developmentand/or differentiation of various cell lineages, such as neural cells,cardiac, hematopoietic cells, skin cells, osteogenic cells and musclecells (e.g., Kuppusamy K T et al., Curr. Mol Med, 2013, 13(5), 757-764;Vidigal J A and Ventura A, Semin Cancer Biol. 2012, 22(5-6), 428-436;Goff L A et al., PLoS One, 2009, 4:e7192; Morin R D et al., Genome Res,2008,18, 610-621; Yoo J K et al., Stem Cells Dev. 2012, 21(11),2049-2057, each of which is herein incorporated by reference in itsentirety). MiRNAs abundant in embryonic stem cells include, but are notlimited to, let-7a-2-3p, let-a-3p, let-7a-5p, let7d-3p, let-7d-5p,miR-103a-2-3p, miR-103a-5p, miR-106b-3p, miR-106b-5p, miR-1246,miR-1275, miR-138-1-3p, miR-138-2-3p, miR-138-5p, miR-154-3p,miR-154-5p, miR-200c-3p, miR-200c-5p, miR-290, miR-301a-3p, miR-301a-5p,miR-302a-3p, miR-302a-5p, miR-302b-3p, miR-302b-5p, miR-302c-3p,miR-302c-5p, miR-302d-3p, miR-302d-5p, miR-302e, miR-367-3p, miR-367-5p,miR-369-3p, miR-369-5p, miR-370, miR-371, miR-373, miR-380-5p,miR-423-3p, miR-423-5p, miR-486-5p, miR-520c-3p, miR-548e, miR-548f,miR-548g-3p, miR-548g-5p, miR-548i, miR-548k, miR-5481, miR-548m,miR-548n, miR-548o-3p, miR-548o-5p, miR-548p, miR-664a-3p, miR-664a-5p,miR-664b-3p, miR-664b-5p, miR-766-3p, miR-766-5p, miR-885-3p,miR-885-5p, miR-93-3p, miR-93-5p, miR-941, miR-96-3p, miR-96-5p,miR-99b-3p and miR-99b-5p. Many predicted novel miRNAs are discovered bydeep sequencing in human embryonic stem cells (e.g., Morin R D et al.,Genome Res, 2008,18, 610-621; Goff L A et al., PLoS One, 2009, 4:e7192;Bar M et al., Stem cells, 2008, 26, 2496-2505, the content of each ofwhich is incorporated herein by reference in its entirety).

In one embodiment, the binding sites of embryonic stem cell specificmiRNAs can be included in or removed from the 3′UTR of a polynucleotideof the disclosure to modulate the development and/or differentiation ofembryonic stem cells, to inhibit the senescence of stem cells in adegenerative condition (e.g. degenerative diseases), or to stimulate thesenescence and apoptosis of stem cells in a disease condition (e.g.cancer stem cells).

Many miRNA expression studies are conducted to profile the differentialexpression of miRNAs in various cancer cells/tissues and other diseases.Some miRNAs are abnormally over-expressed in certain cancer cells andothers are under-expressed. For example, miRNAs are differentiallyexpressed in cancer cells (WO2008/154098, US2013/0059015,US2013/0042333, WO2011/157294); cancer stem cells (US2012/0053224);pancreatic cancers and diseases (US2009/0131348, US2011/0171646,US2010/0286232, U.S. Pat. No. 8,389,210); asthma and inflammation (U.S.Pat. No. 8,415,096); prostate cancer (US2013/0053264); hepatocellularcarcinoma (WO2012/151212, US2012/0329672, WO2008/054828, U.S. Pat. No.8,252,538); lung cancer cells (WO2011/076143, WO2013/033640,WO2009/070653, US2010/0323357); cutaneous T cell lymphoma(WO2013/011378); colorectal cancer cells (WO2011/0281756,WO2011/076142); cancer positive lymph nodes (WO2009/100430,US2009/0263803); nasopharyngeal carcinoma (EP2112235); chronicobstructive pulmonary disease (US2012/0264626, US2013/0053263); thyroidcancer (WO2013/066678); ovarian cancer cells (US2012/0309645,WO2011/095623); breast cancer cells (WO2008/154098, WO2007/081740,US2012/0214699), leukemia and lymphoma (WO2008/073915, US2009/0092974,US2012/0316081, US2012/0283310, WO2010/018563, the content of each ofwhich is incorporated herein by reference in its entirety.)

As a non-limiting example, miRNA binding sites for miRNAs that areover-expressed in certain cancer and/or tumor cells can be removed fromthe 3′UTR of a polynucleotide of the disclosure, restoring theexpression suppressed by the over-expressed miRNAs in cancer cells, thusameliorating the corresponsive biological function, for instance,transcription stimulation and/or repression, cell cycle arrest,apoptosis and cell death. Normal cells and tissues, wherein miRNAsexpression is not up-regulated, will remain unaffected.

miRNA can also regulate complex biological processes such asangiogenesis (e.g., miR-132) (Anand and Cheresh Curr Opin Hematol 201118:171-176). In the polynucleotides of the disclosure, miRNA bindingsites that are involved in such processes can be removed or introduced,in order to tailor the expression of the polynucleotides to biologicallyrelevant cell types or relevant biological processes. In this context,the polynucleotides of the disclosure are defined as auxotrophicpolynucleotides.

In some embodiments, a polynucleotide of the disclosure comprises amiRNA binding site, wherein the miRNA binding site comprises one or morenucleotide sequences selected from Table 7, including one or more copiesof any one or more of the miRNA binding site sequences. In someembodiments, a polynucleotide of the disclosure further comprises atleast one, two, three, four, five, six, seven, eight, nine, ten, or moreof the same or different miRNA binding sites selected from Table 7,including any combination thereof. In some embodiments, the miRNAbinding site binds to miR-142 or is complementary to miR-142. In someembodiments, the miR-142 comprises SEQ ID NO: 720. In some embodiments,the miRNA binding site binds to miR-142-3p or miR-142-5p. In someembodiments, the miR-142-3p binding site comprises SEQ ID NO: 721. Insome embodiments, the miR-142-5p binding site comprises SEQ ID NO: 723.In some embodiments, the miRNA binding site comprises a nucleotidesequence at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 722 or SEQ ID NO: 724.

TABLE 7 miR-142 and miR-142 binding sites SEQ ID NO. DescriptionSequence 720 miR-142 GACAGUGCAGUCACCCAUAAAGUAGAAAGCACUACUAACAGCACUGGAGGGUGUAGUGUUUC CUACUUUAUGGAUGAGUGUACUGUG 721 miR-142-3pUGUAGUGUUUCCUACUUUAUGGA 722 miR-142-3p UCCAUAAAGUAGGAAACACUACAbinding site 723 miR-142-5p CAUAAAGUAGAAAGCACUACU 724 miR-142-5pAGUAGUGCUUUCUACUUUAUG binding site

In some embodiments, a miRNA binding site is inserted in thepolynucleotide of the disclosure in any position of the polynucleotide(e.g., the 5′UTR and/or 3′UTR). In some embodiments, the 5′UTR comprisesa miRNA binding site. In some embodiments, the 3′UTR comprises a miRNAbinding site. In some embodiments, the 5′UTR and the 3′UTR comprise amiRNA binding site. The insertion site in the polynucleotide can beanywhere in the polynucleotide as long as the insertion of the miRNAbinding site in the polynucleotide does not interfere with thetranslation of a functional polypeptide in the absence of thecorresponding miRNA; and in the presence of the miRNA, the insertion ofthe miRNA binding site in the polynucleotide and the binding of themiRNA binding site to the corresponding miRNA are capable of degradingthe polynucleotide or preventing the translation of the polynucleotide.

In some embodiments, a miRNA binding site is inserted in at least about30 nucleotides downstream from the stop codon of an ORF in apolynucleotide of the disclosure comprising the ORF. In someembodiments, a miRNA binding site is inserted in at least about 10nucleotides, at least about 15 nucleotides, at least about 20nucleotides, at least about 25 nucleotides, at least about 30nucleotides, at least about 35 nucleotides, at least about 40nucleotides, at least about 45 nucleotides, at least about 50nucleotides, at least about 55 nucleotides, at least about 60nucleotides, at least about 65 nucleotides, at least about 70nucleotides, at least about 75 nucleotides, at least about 80nucleotides, at least about 85 nucleotides, at least about 90nucleotides, at least about 95 nucleotides, or at least about 100nucleotides downstream from the stop codon of an ORF in a polynucleotideof the disclosure. In some embodiments, a miRNA binding site is insertedin about 10 nucleotides to about 100 nucleotides, about 20 nucleotidesto about 90 nucleotides, about 30 nucleotides to about 80 nucleotides,about 40 nucleotides to about 70 nucleotides, about 50 nucleotides toabout 60 nucleotides, about 45 nucleotides to about 65 nucleotidesdownstream from the stop codon of an ORF in a polynucleotide of thedisclosure.

miRNA gene regulation can be influenced by the sequence surrounding themiRNA such as, but not limited to, the species of the surroundingsequence, the type of sequence (e.g., heterologous, homologous,exogenous, endogenous, or artificial), regulatory elements in thesurrounding sequence and/or structural elements in the surroundingsequence. The miRNA can be influenced by the 5′UTR and/or 3′UTR. As anon-limiting example, a non-human 3′UTR can increase the regulatoryeffect of the miRNA sequence on the expression of a polypeptide ofinterest compared to a human 3′UTR of the same sequence type.

In one embodiment, other regulatory elements and/or structural elementsof the 5′UTR can influence miRNA mediated gene regulation. One exampleof a regulatory element and/or structural element is a structured IRES(Internal Ribosome Entry Site) in the 5′UTR, which is necessary for thebinding of translational elongation factors to initiate proteintranslation. EIF4A2 binding to this secondarily structured element inthe 5′-UTR is necessary for miRNA mediated gene expression (Meijer H Aet al., Science, 2013, 340, 82-85, herein incorporated by reference inits entirety). The polynucleotides of the disclosure can further includethis structured 5′UTR in order to enhance microRNA mediated generegulation.

At least one miRNA binding site can be engineered into the 3′UTR of apolynucleotide of the disclosure. In this context, at least two, atleast three, at least four, at least five, at least six, at least seven,at least eight, at least nine, at least ten, or more miRNA binding sitescan be engineered into a 3′UTR of a polynucleotide of the disclosure.For example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1to 3, 2, or 1 miRNA binding sites can be engineered into the 3′UTR of apolynucleotide of the disclosure. In one embodiment, miRNA binding sitesincorporated into a polynucleotide of the disclosure can be the same orcan be different miRNA sites. A combination of different miRNA bindingsites incorporated into a polynucleotide of the disclosure can includecombinations in which more than one copy of any of the different miRNAsites are incorporated. In another embodiment, miRNA binding sitesincorporated into a polynucleotide of the disclosure can target the sameor different tissues in the body. As a non-limiting example, through theintroduction of tissue-, cell-type-, or disease-specific miRNA bindingsites in the 3′-UTR of a polynucleotide of the disclosure, the degree ofexpression in specific cell types (e.g., hepatocytes, myeloid cells,endothelial cells, cancer cells, etc.) can be reduced.

In one embodiment, a miRNA binding site can be engineered near the 5′terminus of the 3′UTR, about halfway between the 5′ terminus and 3′terminus of the 3′UTR and/or near the 3′ terminus of the 3′UTR in apolynucleotide of the disclosure. As a non-limiting example, a miRNAbinding site can be engineered near the 5′ terminus of the 3′UTR andabout halfway between the 5′ terminus and 3′ terminus of the 3′UTR. Asanother non-limiting example, a miRNA binding site can be engineerednear the 3′ terminus of the 3′UTR and about halfway between the 5′terminus and 3′ terminus of the 3′UTR. As yet another non-limitingexample, a miRNA binding site can be engineered near the 5′ terminus ofthe 3′UTR and near the 3′ terminus of the 3′UTR.

In another embodiment, a 3′UTR can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 miRNA binding sites. The miRNA binding sites can be complementaryto a miRNA, miRNA seed sequence, and/or miRNA sequences flanking theseed sequence.

In one embodiment, a polynucleotide of the disclosure can be engineeredto include more than one miRNA site expressed in different tissues ordifferent cell types of a subject. As a non-limiting example, apolynucleotide of the disclosure can be engineered to include miR-192and miR-122 to regulate expression of the polynucleotide in the liverand kidneys of a subject. In another embodiment, a polynucleotide of thedisclosure can be engineered to include more than one miRNA site for thesame tissue.

In some embodiments, the therapeutic window and or differentialexpression associated with the polypeptide encoded by a polynucleotideof the disclosure can be altered with a miRNA binding site. For example,a polynucleotide encoding a polypeptide that provides a death signal canbe designed to be more highly expressed in cancer cells by virtue of themiRNA signature of those cells. Where a cancer cell expresses a lowerlevel of a particular miRNA, the polynucleotide encoding the bindingsite for that miRNA (or miRNAs) would be more highly expressed. Hence,the polypeptide that provides a death signal triggers or induces celldeath in the cancer cell. Neighboring noncancer cells, harboring ahigher expression of the same miRNA would be less affected by theencoded death signal as the polynucleotide would be expressed at a lowerlevel due to the effects of the miRNA binding to the binding site or“sensor” encoded in the 3′UTR. Conversely, cell survival orcytoprotective signals can be delivered to tissues containing cancer andnon-cancerous cells where a miRNA has a higher expression in the cancercells—the result being a lower survival signal to the cancer cell and alarger survival signal to the normal cell. Multiple polynucleotides canbe designed and administered having different signals based on the useof miRNA binding sites as described herein.

In some embodiments, the expression of a polynucleotide of thedisclosure can be controlled by incorporating at least one sensorsequence in the polynucleotide and formulating the polynucleotide foradministration. As a non-limiting example, a polynucleotide of thedisclosure can be targeted to a tissue or cell by incorporating a miRNAbinding site and formulating the polynucleotide in a lipid nanoparticlecomprising a cationic lipid, including any of the lipids describedherein.

A polynucleotide of the disclosure can be engineered for more targetedexpression in specific tissues, cell types, or biological conditionsbased on the expression patterns of miRNAs in the different tissues,cell types, or biological conditions. Through introduction oftissue-specific miRNA binding sites, a polynucleotide of the disclosurecan be designed for optimal protein expression in a tissue or cell, orin the context of a biological condition.

In some embodiments, a polynucleotide of the disclosure can be designedto incorporate miRNA binding sites that either have 100% identity toknown miRNA seed sequences or have less than 100% identity to miRNA seedsequences. In some embodiments, a polynucleotide of the disclosure canbe designed to incorporate miRNA binding sites that have at least: 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity toknown miRNA seed sequences. The miRNA seed sequence can be partiallymutated to decrease miRNA binding affinity and as such result in reduceddownmodulation of the polynucleotide. In essence, the degree of match ormis-match between the miRNA binding site and the miRNA seed can act as arheostat to more finely tune the ability of the miRNA to modulateprotein expression. In addition, mutation in the non-seed region of amiRNA binding site can also impact the ability of a miRNA to modulateprotein expression.

In one embodiment, a miRNA sequence can be incorporated into the loop ofa stem loop.

In another embodiment, a miRNA seed sequence can be incorporated in theloop of a stem loop and a miRNA binding site can be incorporated intothe 5′ or 3′ stem of the stem loop.

In one embodiment, a translation enhancer element (TEE) can beincorporated on the 5′end of the stem of a stem loop and a miRNA seedcan be incorporated into the stem of the stem loop. In anotherembodiment, a TEE can be incorporated on the 5′ end of the stem of astem loop, a miRNA seed can be incorporated into the stem of the stemloop and a miRNA binding site can be incorporated into the 3′ end of thestem or the sequence after the stem loop. The miRNA seed and the miRNAbinding site can be for the same and/or different miRNA sequences.

In one embodiment, the incorporation of a miRNA sequence and/or a TEEsequence changes the shape of the stem loop region which can increaseand/or decrease translation. (see e.g, Kedde et al., “A Pumilio-inducedRNA structure switch in p27-3′UTR controls miR-221 and miR-22accessibility.” Nature Cell Biology. 2010, incorporated herein byreference in its entirety).

In one embodiment, the 5′-UTR of a polynucleotide of the disclosure cancomprise at least one miRNA sequence. The miRNA sequence can be, but isnot limited to, a 19 or 22 nucleotide sequence and/or a miRNA sequencewithout the seed.

In one embodiment the miRNA sequence in the 5′UTR can be used tostabilize a polynucleotide of the disclosure described herein.

In another embodiment, a miRNA sequence in the 5′UTR of a polynucleotideof the disclosure can be used to decrease the accessibility of the siteof translation initiation such as, but not limited to a start codon.See, e.g., Matsuda et al., PLoS One. 2010 11(5):e15057; incorporatedherein by reference in its entirety, which used antisense locked nucleicacid (LNA) oligonucleotides and exon-junction complexes (EJCs) around astart codon (−4 to +37 where the A of the AUG codons is +1) in order todecrease the accessibility to the first start codon (AUG). Matsudashowed that altering the sequence around the start codon with an LNA orEJC affected the efficiency, length and structural stability of apolynucleotide. A polynucleotide of the disclosure can comprise a miRNAsequence, instead of the LNA or EJC sequence described by Matsuda et al,near the site of translation initiation in order to decrease theaccessibility to the site of translation initiation. The site oftranslation initiation can be prior to, after or within the miRNAsequence. As a non-limiting example, the site of translation initiationcan be located within a miRNA sequence such as a seed sequence orbinding site. As another non-limiting example, the site of translationinitiation can be located within a miR-122 sequence such as the seedsequence or the mir-122 binding site.

In some embodiments, a polynucleotide of the disclosure can include atleast one miRNA in order to dampen the antigen presentation by antigenpresenting cells. The miRNA can be the complete miRNA sequence, themiRNA seed sequence, the miRNA sequence without the seed, or acombination thereof. As a non-limiting example, a miRNA incorporatedinto a polynucleotide of the disclosure can be specific to thehematopoietic system. As another non-limiting example, a miRNAincorporated into a polynucleotide of the disclosure to dampen antigenpresentation is miR-142-3p.

In some embodiments, a polynucleotide of the disclosure can include atleast one miRNA in order to dampen expression of the encoded polypeptidein a tissue or cell of interest. As a non-limiting example, apolynucleotide of the disclosure can include at least one miR-122binding site in order to dampen expression of an encoded polypeptide ofinterest in the liver. As another non-limiting example a polynucleotideof the disclosure can include at least one miR-142-3p binding site,miR-142-3p seed sequence, miR-142-3p binding site without the seed,miR-142-5p binding site, miR-142-5p seed sequence, miR-142-5p bindingsite without the seed, miR-146 binding site, miR-146 seed sequenceand/or miR-146 binding site without the seed sequence.

In some embodiments, a polynucleotide of the disclosure can comprise atleast one miRNA binding site in the 3′UTR in order to selectivelydegrade mRNA therapeutics in the immune cells to subdue unwantedimmunogenic reactions caused by therapeutic delivery. As a non-limitingexample, the miRNA binding site can make a polynucleotide of thedisclosure more unstable in antigen presenting cells. Non-limitingexamples of these miRNAs include mir-142-5p, mir-142-3p, mir-146a-5p,and mir-146-3p.

In one embodiment, a polynucleotide of the disclosure comprises at leastone miRNA sequence in a region of the polynucleotide that can interactwith a RNA binding protein.

In some embodiments, the polynucleotide of the disclosure (e.g., a RNA,e.g., a mRNA) comprising (i) a sequence-optimized nucleotide sequence(e.g., an ORF) encoding a MCM polypeptide (e.g., the wild-type sequence,functional fragment, or variant thereof) and (ii) a miRNA binding site(e.g., a miRNA binding site that binds to miR-142).

In some embodiments, the polynucleotide of the disclosure comprises auracil-modified sequence encoding a MCM polypeptide disclosed herein anda miRNA binding site disclosed herein, e.g., a miRNA binding site thatbinds to miR-142. In some embodiments, the uracil-modified sequenceencoding a MCM polypeptide comprises at least one chemically modifiednucleobase, e.g., 5-methoxyuracil. In some embodiments, at least 95% ofa type of nucleobase (e.g., uracil) in a uracil-modified sequenceencoding a MCM polypeptide of the disclosure are modified nucleobases.In some embodiments, at least 95% of uracil in a uracil-modifiedsequence encoding a MCM polypeptide is 5-methoxyuridine. In someembodiments, the polynucleotide comprising a nucleotide sequenceencoding a MCM polypeptide disclosed herein and a miRNA binding site isformulated with a delivery agent, e.g., a compound having the Formula(I), e.g., any of Compounds 1-147.

3′ UTR and the AU Rich Elements

The disclosure also includes a polynucleotide that comprises both one ormore 3′ untranslated regions as well as the polynucleotide describedherein, i.e., a polynucleotide comprising an ORF encoding an MCMpolypeptide.

Natural or wild type 3′ UTRs are known to have stretches of Adenosinesand Uridines embedded in them. These AU rich signatures are particularlyprevalent in genes with high rates of turnover. Based on their sequencefeatures and functional properties, the AU rich elements (AREs) can beseparated into three classes (Chen et al, 1995): Class I AREs containseveral dispersed copies of an AUUUA motif within U-rich regions. C-Mycand MyoD contain class I AREs. Class II AREs possess two or moreoverlapping UUAUUUA(U/A)(U/A) nonamers. Molecules containing this typeof AREs include GM-CSF and TNF-a. Class III ARES are less well defined.These U rich regions do not contain an AUUUA motif. c-Jun and Myogeninare two well-studied examples of this class. Most proteins binding tothe AREs are known to destabilize the messenger, whereas members of theELAV family, most notably HuR, have been documented to increase thestability of mRNA. HuR binds to AREs of all the three classes.Engineering the HuR specific binding sites into the 3′ UTR of nucleicacid molecules will lead to HuR binding and thus, stabilization of themessage in vivo.

Introduction, removal or modification of 3′ UTR AU rich elements (AREs)can be used to modulate the stability of polynucleotides of thedisclosure. When engineering specific polynucleotides, one or morecopies of an ARE can be introduced to make polynucleotides of thedisclosure less stable and thereby curtail translation and decreaseproduction of the resultant protein. Likewise, AREs can be identifiedand removed or mutated to increase the intracellular stability and thusincrease translation and production of the resultant protein.Transfection experiments can be conducted in relevant cell lines, usingpolynucleotides of the disclosure and protein production can be assayedat various time points post-transfection. For example, cells can betransfected with different ARE-engineering molecules and by using anELISA kit to the relevant protein and assaying protein produced at 6hour, 12 hour, 24 hour, 48 hour, and 7 days post-transfection.

Regions Having a 5′ Cap

The disclosure also includes a polynucleotide that comprises both a 5′Cap and the polynucleotide described herein, i.e., a polynucleotidecomprising an ORF encoding an MCM polypeptide.

The 5′ cap structure of a natural mRNA is involved in nuclear export,increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP),which is responsible for mRNA stability in the cell and translationcompetency through the association of CBP with poly(A) binding proteinto form the mature cyclic mRNA species. The cap further assists theremoval of 5′ proximal introns during mRNA splicing.

Endogenous mRNA molecules can be 5′-end capped generating a5′-ppp-5′-triphosphate linkage between a terminal guanosine cap residueand the 5′-terminal transcribed sense nucleotide of the mRNA molecule.This 5′-guanylate cap can then be methylated to generate anN7-methyl-guanylate residue. The ribose sugars of the terminal and/oranteterminal transcribed nucleotides of the 5′ end of the mRNA canoptionally also be 2′-O-methylated. 5′-decapping through hydrolysis andcleavage of the guanylate cap structure can target a nucleic acidmolecule, such as an mRNA molecule, for degradation.

In some embodiments, polynucleotides can be designed to incorporate acap moiety. Modifications to the polynucleotides of the presentdisclosure can generate a non-hydrolyzable cap structure preventingdecapping and thus increasing mRNA half-life. Because cap structurehydrolysis requires cleavage of 5′-ppp-5′ phosphorodiester linkages,modified nucleotides can be used during the capping reaction. Forexample, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich,Mass.) can be used with α-thio-guanosine nucleotides according to themanufacturer's instructions to create a phosphorothioate linkage in the5′-ppp-5′ cap. Additional modified guanosine nucleotides can be usedsuch as α-methyl-phosphonate and seleno-phosphate nucleotides.

Additional modifications include, but are not limited to,2′-O-methylation of the ribose sugars of 5′-terminal and/or5′-anteterminal nucleotides of the polynucleotide (as mentioned above)on the 2′-hydroxyl group of the sugar ring. Multiple distinct 5′-capstructures can be used to generate the 5′-cap of a nucleic acidmolecule, such as a polynucleotide that functions as an mRNA molecule.

Cap analogs, which herein are also referred to as synthetic cap analogs,chemical caps, chemical cap analogs, or structural or functional capanalogs, differ from natural (i.e., endogenous, wild-type orphysiological) 5′-caps in their chemical structure, while retaining capfunction. Cap analogs can be chemically (i.e., non-enzymatically) orenzymatically synthesized and/or linked to the polynucleotides of thedisclosure.

For example, the Anti-Reverse Cap Analog (ARCA) cap contains twoguanines linked by a 5′-5′-triphosphate group, wherein one guaninecontains an N7 methyl group as well as a 3′-O-methyl group (i.e.,N7,3′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine (m⁷G-3′mppp-G;which can equivalently be designated 3′ O-Me-m7G(5′)ppp(5′)G). The 3′-0atom of the other, unmodified, guanine becomes linked to the 5′-terminalnucleotide of the capped polynucleotide. The N7- and 3′-O-methylatedguanine provides the terminal moiety of the capped polynucleotide.

Another exemplary cap is mCAP, which is similar to ARCA but has a2′-O-methyl group on guanosine (i.e.,N7,2′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine, m⁷Gm-ppp-G).

In some embodiments, the cap is a dinucleotide cap analog. As anon-limiting example, the dinucleotide cap analog can be modified atdifferent phosphate positions with a boranophosphate group or aphophoroselenoate group such as the dinucleotide cap analogs describedin U.S. Pat. No. 8,519,110, the contents of which are hereinincorporated by reference in its entirety.

In another embodiment, the cap is a cap analog is aN7-(4-chlorophenoxyethyl) substituted dicucleotide form of a cap analogknown in the art and/or described herein. Non-limiting examples of aN7-(4-chlorophenoxyethyl) substituted dicucleotide form of a cap analoginclude a N7-(4-chlorophenoxyethyl)-G(5′)ppp(5′)G and aN7-(4-chlorophenoxyethyl)-m^(3′-O)G(5′)ppp(5′)G cap analog (See, e.g.,the various cap analogs and the methods of synthesizing cap analogsdescribed in Kore et al. Bioorganic & Medicinal Chemistry 201321:4570-4574; the contents of which are herein incorporated by referencein its entirety). In another embodiment, a cap analog of the presentdisclosure is a 4-chloro/bromophenoxyethyl analog.

While cap analogs allow for the concomitant capping of a polynucleotideor a region thereof, in an in vitro transcription reaction, up to 20% oftranscripts can remain uncapped. This, as well as the structuraldifferences of a cap analog from an endogenous 5′-cap structures ofnucleic acids produced by the endogenous, cellular transcriptionmachinery, can lead to reduced translational competency and reducedcellular stability.

Polynucleotides of the disclosure can also be capped post-manufacture(whether IVT or chemical synthesis), using enzymes, in order to generatemore authentic 5′-cap structures. As used herein, the phrase “moreauthentic” refers to a feature that closely mirrors or mimics, eitherstructurally or functionally, an endogenous or wild type feature. Thatis, a “more authentic” feature is better representative of anendogenous, wild-type, natural or physiological cellular function and/orstructure as compared to synthetic features or analogs, etc., of theprior art, or which outperforms the corresponding endogenous, wild-type,natural or physiological feature in one or more respects. Non-limitingexamples of more authentic 5′cap structures of the present disclosureare those that, among other things, have enhanced binding of cap bindingproteins, increased half-life, reduced susceptibility to 5′endonucleases and/or reduced 5′decapping, as compared to synthetic 5′capstructures known in the art (or to a wild-type, natural or physiological5′cap structure). For example, recombinant Vaccinia Virus Capping Enzymeand recombinant 2′-O-methyltransferase enzyme can create a canonical5′-5′-triphosphate linkage between the 5′-terminal nucleotide of apolynucleotide and a guanine cap nucleotide wherein the cap guaninecontains an N7 methylation and the 5′-terminal nucleotide of the mRNAcontains a 2′-O-methyl. Such a structure is termed the Cap1 structure.This cap results in a higher translational-competency and cellularstability and a reduced activation of cellular pro-inflammatorycytokines, as compared, e.g., to other 5′cap analog structures known inthe art. Cap structures include, but are not limited to,7mG(5′)ppp(5′)N,pN2p (cap 0), 7mG(5′)ppp(5′)NlmpNp (cap 1), and7mG(5′)-ppp(5′)NlmpN2mp (cap 2).

As a non-limiting example, capping chimeric polynucleotidespost-manufacture can be more efficient as nearly 100% of the chimericpolynucleotides can be capped. This is in contrast to ˜80% when a capanalog is linked to a chimeric polynucleotide in the course of an invitro transcription reaction.

According to the present disclosure, 5′ terminal caps can includeendogenous caps or cap analogs. According to the present disclosure, a5′ terminal cap can comprise a guanine analog. Useful guanine analogsinclude, but are not limited to, inosine, N1-methyl-guanosine,2′fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine,2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

Poly-A Tails

The disclosure also includes a polynucleotide that comprises both apoly-A tail and the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide.

During RNA processing, a long chain of adenine nucleotides (poly-A tail)can be added to a polynucleotide such as an mRNA molecule in order toincrease stability. Immediately after transcription, the 3′ end of thetranscript can be cleaved to free a 3′ hydroxyl. Then poly-A polymeraseadds a chain of adenine nucleotides to the RNA. The process, calledpolyadenylation, adds a poly-A tail that can be between, for example,approximately 80 to approximately 250 residues long, includingapproximately 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 210, 220, 230, 240 or 250 residues long.

PolyA tails can also be added after the construct is exported from thenucleus.

According to the present disclosure, terminal groups on the poly A tailcan be incorporated for stabilization. Polynucleotides of the presentdisclosure can include des-3′ hydroxyl tails. They can also includestructural moieties or 2′-Omethyl modifications as taught by Junjie Li,et al. (Current Biology, Vol. 15, 1501-1507, Aug. 23, 2005, the contentsof which are incorporated herein by reference in its entirety).

The polynucleotides of the present disclosure can be designed to encodetranscripts with alternative polyA tail structures including histonemRNA. According to Norbury, “Terminal uridylation has also been detectedon human replication-dependent histone mRNAs. The turnover of thesemRNAs is thought to be important for the prevention of potentially toxichistone accumulation following the completion or inhibition ofchromosomal DNA replication. These mRNAs are distinguished by their lackof a 3′ poly(A) tail, the function of which is instead assumed by astable stem-loop structure and its cognate stem-loop binding protein(SLBP); the latter carries out the same functions as those of PABP onpolyadenylated mRNAs” (Norbury, “Cytoplasmic RNA: a case of the tailwagging the dog,” Nature Reviews Molecular Cell Biology; AOP, publishedonline 29 Aug. 2013; doi:10.1038/nrm3645) the contents of which areincorporated herein by reference in its entirety.

Unique poly-A tail lengths provide certain advantages to thepolynucleotides of the present disclosure.

Generally, the length of a poly-A tail, when present, is greater than 30nucleotides in length. In another embodiment, the poly-A tail is greaterthan 35 nucleotides in length (e.g., at least or greater than about 35,40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300,350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300,1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000nucleotides). In some embodiments, the polynucleotide or region thereofincludes from about 30 to about 3,000 nucleotides (e.g., from 30 to 50,from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500to 750, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to2,000, from 1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000,from 1,500 to 2,500, from 1,500 to 3,000, from 2,000 to 3,000, from2,000 to 2,500, and from 2,500 to 3,000).

In some embodiments, the poly-A tail is designed relative to the lengthof the overall polynucleotide or the length of a particular region ofthe polynucleotide. This design can be based on the length of a codingregion, the length of a particular feature or region or based on thelength of the ultimate product expressed from the polynucleotides.

In this context, the poly-A tail can be 10, 20, 30, 40, 50, 60, 70, 80,90, or 100% greater in length than the polynucleotide or featurethereof. The poly-A tail can also be designed as a fraction of thepolynucleotides to which it belongs. In this context, the poly-A tailcan be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the totallength of the construct, a construct region or the total length of theconstruct minus the poly-A tail. Further, engineered binding sites andconjugation of polynucleotides for Poly-A binding protein can enhanceexpression.

Additionally, multiple distinct polynucleotides can be linked togethervia the PABP (Poly-A binding protein) through the 3′-end using modifiednucleotides at the 3′-terminus of the poly-A tail. Transfectionexperiments can be conducted in relevant cell lines at and proteinproduction can be assayed by ELISA at 12 hr, 24 hr, 48 hr, 72 hr and day7 post-transfection.

In some embodiments, the polynucleotides of the present disclosure aredesigned to include a polyA-G Quartet region. The G-quartet is a cyclichydrogen bonded array of four guanine nucleotides that can be formed byG-rich sequences in both DNA and RNA. In this embodiment, the G-quartetis incorporated at the end of the poly-A tail. The resultantpolynucleotide is assayed for stability, protein production and otherparameters including half-life at various time points. It has beendiscovered that the polyA-G quartet results in protein production froman mRNA equivalent to at least 75% of that seen using a poly-A tail of120 nucleotides alone.

Start Codon Region

The disclosure also includes a polynucleotide that comprises both astart codon region and the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide. In someembodiments, the polynucleotides of the present disclosure can haveregions that are analogous to or function like a start codon region.

In some embodiments, the translation of a polynucleotide can initiate ona codon that is not the start codon AUG. Translation of thepolynucleotide can initiate on an alternative start codon such as, butnot limited to, ACG, AGG, AAG, CTG/CUG, GTG/GUG, ATA/AUA, ATT/AUU,TTG/UUG (see Touriol et al. Biology of the Cell 95 (2003) 169-178 andMatsuda and Mauro PLoS ONE, 2010 5:11; the contents of each of which areherein incorporated by reference in its entirety). As a non-limitingexample, the translation of a polynucleotide begins on the alternativestart codon ACG. As another non-limiting example, polynucleotidetranslation begins on the alternative start codon CTG or CUG. As yetanother non-limiting example, the translation of a polynucleotide beginson the alternative start codon GTG or GUG.

Nucleotides flanking a codon that initiates translation such as, but notlimited to, a start codon or an alternative start codon, are known toaffect the translation efficiency, the length and/or the structure ofthe polynucleotide. (See, e.g., Matsuda and Mauro PLoS ONE, 2010 5:11;the contents of which are herein incorporated by reference in itsentirety). Masking any of the nucleotides flanking a codon thatinitiates translation can be used to alter the position of translationinitiation, translation efficiency, length and/or structure of apolynucleotide.

In some embodiments, a masking agent can be used near the start codon oralternative start codon in order to mask or hide the codon to reduce theprobability of translation initiation at the masked start codon oralternative start codon. Non-limiting examples of masking agents includeantisense locked nucleic acids (LNA) polynucleotides and exon-junctioncomplexes (EJCs) (See, e.g., Matsuda and Mauro describing masking agentsLNA polynucleotides and EJCs (PLoS ONE, 2010 5:11); the contents ofwhich are herein incorporated by reference in its entirety).

In another embodiment, a masking agent can be used to mask a start codonof a polynucleotide in order to increase the likelihood that translationwill initiate on an alternative start codon.

In some embodiments, a masking agent can be used to mask a first startcodon or alternative start codon in order to increase the chance thattranslation will initiate on a start codon or alternative start codondownstream to the masked start codon or alternative start codon.

In some embodiments, a start codon or alternative start codon can belocated within a perfect complement for a miR binding site. The perfectcomplement of a miR binding site can help control the translation,length and/or structure of the polynucleotide similar to a maskingagent. As a non-limiting example, the start codon or alternative startcodon can be located in the middle of a perfect complement for a miR-122binding site. The start codon or alternative start codon can be locatedafter the first nucleotide, second nucleotide, third nucleotide, fourthnucleotide, fifth nucleotide, sixth nucleotide, seventh nucleotide,eighth nucleotide, ninth nucleotide, tenth nucleotide, eleventhnucleotide, twelfth nucleotide, thirteenth nucleotide, fourteenthnucleotide, fifteenth nucleotide, sixteenth nucleotide, seventeenthnucleotide, eighteenth nucleotide, nineteenth nucleotide, twentiethnucleotide or twenty-first nucleotide.

In another embodiment, the start codon of a polynucleotide can beremoved from the polynucleotide sequence in order to have thetranslation of the polynucleotide begin on a codon that is not the startcodon. Translation of the polynucleotide can begin on the codonfollowing the removed start codon or on a downstream start codon or analternative start codon. In a non-limiting example, the start codon ATGor AUG is removed as the first 3 nucleotides of the polynucleotidesequence in order to have translation initiate on a downstream startcodon or alternative start codon. The polynucleotide sequence where thestart codon was removed can further comprise at least one masking agentfor the downstream start codon and/or alternative start codons in orderto control or attempt to control the initiation of translation, thelength of the polynucleotide and/or the structure of the polynucleotide.

Stop Codon Region

The disclosure also includes a polynucleotide that comprises both a stopcodon region and the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide. In someembodiments, the polynucleotides of the present disclosure can includeat least two stop codons before the 3′ untranslated region (UTR). Thestop codon can be selected from TGA, TAA and TAG. In some embodiments,the polynucleotides of the present disclosure include the stop codon TGAand one additional stop codon. In a further embodiment the addition stopcodon can be TAA. In another embodiment, the polynucleotides of thepresent disclosure include three stop codons.

Insertions and Substitutions

The disclosure also includes a polynucleotide that comprises insertionsand/or substitutions in the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide.

In some embodiments, the 5′UTR of the polynucleotide can be replaced bythe insertion of at least one region and/or string of nucleosides of thesame base. The region and/or string of nucleotides can include, but isnot limited to, at least 3, at least 4, at least 5, at least 6, at least7 or at least 8 nucleotides and the nucleotides can be natural and/orunnatural. As a non-limiting example, the group of nucleotides caninclude 5-8 adenine, cytosine, thymine, a string of any of the othernucleotides disclosed herein and/or combinations thereof.

In some embodiments, the 5′UTR of the polynucleotide can be replaced bythe insertion of at least two regions and/or strings of nucleotides oftwo different bases such as, but not limited to, adenine, cytosine,thymine, any of the other nucleotides disclosed herein and/orcombinations thereof. For example, the 5′UTR can be replaced byinserting 5-8 adenine bases followed by the insertion of 5-8 cytosinebases. In another example, the 5′UTR can be replaced by inserting 5-8cytosine bases followed by the insertion of 5-8 adenine bases.

In some embodiments, the polynucleotide can include at least onesubstitution and/or insertion downstream of the transcription start sitethat can be recognized by an RNA polymerase. As a non-limiting example,at least one substitution and/or insertion can occur downstream of thetranscription start site by substituting at least one nucleic acid inthe region just downstream of the transcription start site (such as, butnot limited to, +1 to +6). Changes to region of nucleotides justdownstream of the transcription start site can affect initiation rates,increase apparent nucleotide triphosphate (NTP) reaction constantvalues, and increase the dissociation of short transcripts from thetranscription complex curing initial transcription (Brieba et al,Biochemistry (2002) 41: 5144-5149; herein incorporated by reference inits entirety). The modification, substitution and/or insertion of atleast one nucleoside can cause a silent mutation of the sequence or cancause a mutation in the amino acid sequence.

In some embodiments, the polynucleotide can include the substitution ofat least 1, at least 2, at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, at least 11, at least12 or at least 13 guanine bases downstream of the transcription startsite.

In some embodiments, the polynucleotide can include the substitution ofat least 1, at least 2, at least 3, at least 4, at least 5 or at least 6guanine bases in the region just downstream of the transcription startsite. As a non-limiting example, if the nucleotides in the region areGGGAGA, the guanine bases can be substituted by at least 1, at least 2,at least 3 or at least 4 adenine nucleotides. In another non-limitingexample, if the nucleotides in the region are GGGAGA the guanine basescan be substituted by at least 1, at least 2, at least 3 or at least 4cytosine bases. In another non-limiting example, if the nucleotides inthe region are GGGAGA the guanine bases can be substituted by at least1, at least 2, at least 3 or at least 4 thymine, and/or any of thenucleotides described herein.

In some embodiments, the polynucleotide can include at least onesubstitution and/or insertion upstream of the start codon. For thepurpose of clarity, one of skill in the art would appreciate that thestart codon is the first codon of the protein coding region whereas thetranscription start site is the site where transcription begins. Thepolynucleotide can include, but is not limited to, at least 1, at least2, at least 3, at least 4, at least 5, at least 6, at least 7 or atleast 8 substitutions and/or insertions of nucleotide bases. Thenucleotide bases can be inserted or substituted at 1, at least 1, atleast 2, at least 3, at least 4 or at least 5 locations upstream of thestart codon. The nucleotides inserted and/or substituted can be the samebase (e.g., all A or all C or all T or all G), two different bases(e.g., A and C, A and T, or C and T), three different bases (e.g., A, Cand T or A, C and T) or at least four different bases. As a non-limitingexample, the guanine base upstream of the coding region in thepolynucleotide can be substituted with adenine, cytosine, thymine, orany of the nucleotides described herein. In another non-limiting examplethe substitution of guanine bases in the polynucleotide can be designedso as to leave one guanine base in the region downstream of thetranscription start site and before the start codon (see Esvelt et al.Nature (2011) 472(7344):499-503; the contents of which is hereinincorporated by reference in its entirety). As a non-limiting example,at least 5 nucleotides can be inserted at 1 location downstream of thetranscription start site but upstream of the start codon and the atleast 5 nucleotides can be the same base type.

IV. METHODS OF MAKING POLYNUCLEOTIDES

The present disclosure also provides methods for making a polynucleotidedisclosed herein or a complement thereof. In some aspects, apolynucleotide (e.g., an mRNA) disclosed herein, and encoding an MCMpolypeptide or a functional fragment thereof, can be constructed usingin vitro transcription. In other aspects, a polynucleotide (e.g., anmRNA) disclosed herein, and encoding an MCM polypeptide or a functionalfragment thereof, can be constructed by chemical synthesis using anoligonucleotide synthesizer. In other aspects, a polynucleotide (e.g.,an mRNA) disclosed herein, and encoding an MCM polypeptide or afunctional fragment thereof is made by using a host cell. In certainaspects, a polynucleotide (e.g., an mRNA) disclosed herein, and encodingan MCM polypeptide or a functional fragment thereof is made by one ormore combination of the IVT, chemical synthesis, host cell expression,or any other methods known in the art.

Naturally occurring nucleosides, non-naturally occurring nucleosides, orcombinations thereof, can totally or partially naturally replaceoccurring nucleosides present in the candidate nucleotide sequence andcan be incorporated into a sequence-optimized nucleotide sequence (e.g.,an mRNA) encoding an MCM polypeptide. The resultant mRNAs can then beexamined for their ability to produce protein and/or produce atherapeutic outcome.

In Vitro Transcription-Enzymatic Synthesis

A polynucleotide disclosed herein can be transcribed using an in vitrotranscription (IVT) system. The system typically comprises atranscription buffer, nucleotide triphosphates (NTPs), an RNaseinhibitor and a polymerase. The NTPs can be selected from, but are notlimited to, those described herein including natural and unnatural(modified) NTPs. The polymerase can be selected from, but is not limitedto, T7 RNA polymerase, T3 RNA polymerase and mutant polymerases such as,but not limited to, polymerases able to incorporate modified nucleicacids. See U.S. Publ. No. US20130259923, which is herein incorporated byreference in its entirety.

The IVT system typically comprises a transcription buffer, nucleotidetriphosphates (NTPs), an RNase inhibitor and a polymerase. The NTPs canbe selected from, but are not limited to, those described hereinincluding natural and unnatural (modified) NTPs. The polymerase can beselected from, but is not limited to, T7 RNA polymerase, T3 RNApolymerase and mutant polymerases such as, but not limited to,polymerases able to incorporate polynucleotides disclosed herein.

Any number of RNA polymerases or variants can be used in the synthesisof the polynucleotides of the present disclosure.

RNA polymerases can be modified by inserting or deleting amino acids ofthe RNA polymerase sequence. As a non-limiting example, the RNApolymerase can be modified to exhibit an increased ability toincorporate a 2′-modified nucleotide triphosphate compared to anunmodified RNA polymerase (see International Publication WO2008078180and U.S. Pat. No. 8,101,385; herein incorporated by reference in theirentireties).

Variants can be obtained by evolving an RNA polymerase, optimizing theRNA polymerase amino acid and/or nucleic acid sequence and/or by usingother methods known in the art. As a non-limiting example, T7 RNApolymerase variants can be evolved using the continuous directedevolution system set out by Esvelt et al. (Nature (2011)472(7344):499-503; herein incorporated by reference in its entirety)where clones of T7 RNA polymerase can encode at least one mutation suchas, but not limited to, lysine at position 93 substituted for threonine(K93T), I4M, A7T, E63V, V64D, A65E, D66Y, T76N, C125R, S128R, A136T,N165S, G175R, H176L, Y178H, F182L, L196F, G198V, D208Y, E222K, S228A,Q239R, T243N, G259D, M2671, G280C, H300R, D351A, A354S, E356D, L360P,A383V, Y385C, D388Y, S397R, M401T, N410S, K450R, P451T, G452V, E484A,H523L, H524N, G542V, E565K, K577E, K577M, N601S, S684Y, L699I, K713E,N748D, Q754R, E775K, A827V, D851N or L864F. As another non-limitingexample, T7 RNA polymerase variants can encode at least mutation asdescribed in U.S. Pub. Nos. 20100120024 and 20070117112; hereinincorporated by reference in their entireties. Variants of RNApolymerase can also include, but are not limited to, substitutionalvariants, conservative amino acid substitution, insertional variants,deletional variants and/or covalent derivatives.

In one aspect, the polynucleotide can be designed to be recognized bythe wild type or variant RNA polymerases. In doing so, thepolynucleotide can be modified to contain sites or regions of sequencechanges from the wild type or parent chimeric polynucleotide.

Polynucleotide or nucleic acid synthesis reactions can be carried out byenzymatic methods utilizing polymerases. Polymerases catalyze thecreation of phosphodiester bonds between nucleotides in a polynucleotideor nucleic acid chain. Currently known DNA polymerases can be dividedinto different families based on amino acid sequence comparison andcrystal structure analysis. DNA polymerase I (pol I) or A polymerasefamily, including the Klenow fragments of E. Coli, Bacillus DNApolymerase I, Thermus aquaticus (Taq) DNA polymerases, and the T7 RNAand DNA polymerases, is among the best studied of these families.Another large family is DNA polymerase a (pol a) or B polymerase family,including all eukaryotic replicating DNA polymerases and polymerasesfrom phages T4 and RB69. Although they employ similar catalyticmechanism, these families of polymerases differ in substratespecificity, substrate analog-incorporating efficiency, degree and ratefor primer extension, mode of DNA synthesis, exonuclease activity, andsensitivity against inhibitors.

DNA polymerases are also selected based on the optimum reactionconditions they require, such as reaction temperature, pH, and templateand primer concentrations. Sometimes a combination of more than one DNApolymerases is employed to achieve the desired DNA fragment size andsynthesis efficiency. For example, Cheng et al. increase pH, addglycerol and dimethyl sulfoxide, decrease denaturation times, increaseextension times, and utilize a secondary thermostable DNA polymerasethat possesses a 3′ to 5′ exonuclease activity to effectively amplifylong targets from cloned inserts and human genomic DNA. (Cheng et al.,PNAS, Vol. 91, 5695-5699 (1994), the contents of which are incorporatedherein by reference in their entirety). RNA polymerases frombacteriophage T3, T7, and SP6 have been widely used to prepare RNAs forbiochemical and biophysical studies. RNA polymerases, capping enzymes,and poly-A polymerases are disclosed in the co-pending InternationalPublication No. WO2014028429, the contents of which are incorporatedherein by reference in their entirety.

In one aspect, the RNA polymerase which can be used in the synthesis ofthe polynucleotides described herein is a Syn5 RNA polymerase. (see Zhuet al. Nucleic Acids Research 2013, the contents of which is hereinincorporated by reference in its entirety). The Syn5 RNA polymerase wasrecently characterized from marine cyanophage Syn5 by Zhu et al. wherethey also identified the promoter sequence (see Zhu et al. Nucleic AcidsResearch 2013, the contents of which is herein incorporated by referencein its entirety). Zhu et al. found that Syn5 RNA polymerase catalyzedRNA synthesis over a wider range of temperatures and salinity ascompared to T7 RNA polymerase. Additionally, the requirement for theinitiating nucleotide at the promoter was found to be less stringent forSyn5 RNA polymerase as compared to the T7 RNA polymerase making Syn5 RNApolymerase promising for RNA synthesis.

In one aspect, a Syn5 RNA polymerase can be used in the synthesis of thepolynucleotides described herein. As a non-limiting example, a Syn5 RNApolymerase can be used in the synthesis of the polynucleotide requiringa precise 3′-termini.

In one aspect, a Syn5 promoter can be used in the synthesis of thepolynucleotides. As a non-limiting example, the Syn5 promoter can be5′-ATTGGGCACCCGTAAGGG-3′ as described by Zhu et al. (Nucleic AcidsResearch 2013, the contents of which is herein incorporated by referencein its entirety).

In one aspect, a Syn5 RNA polymerase can be used in the synthesis ofpolynucleotides comprising at least one chemical modification describedherein and/or known in the art. (see e.g., the incorporation ofpseudo-UTP and 5Me-CTP described in Zhu et al. Nucleic Acids Research2013, the contents of which is herein incorporated by reference in itsentirety).

In one aspect, the polynucleotides described herein can be synthesizedusing a Syn5 RNA polymerase which has been purified using modified andimproved purification procedure described by Zhu et al. (Nucleic AcidsResearch 2013, the contents of which is herein incorporated by referencein its entirety).

Various tools in genetic engineering are based on the enzymaticamplification of a target gene which acts as a template. For the studyof sequences of individual genes or specific regions of interest andother research needs, it is necessary to generate multiple copies of atarget gene from a small sample of polynucleotides or nucleic acids.Such methods can be applied in the manufacture of the polynucleotides ofthe disclosure.

Polymerase chain reaction (PCR) has wide applications in rapidamplification of a target gene, as well as genome mapping andsequencing. The key components for synthesizing DNA comprise target DNAmolecules as a template, primers complementary to the ends of target DNAstrands, deoxynucleoside triphosphates (dNTPs) as building blocks, and aDNA polymerase. As PCR progresses through denaturation, annealing andextension steps, the newly produced DNA molecules can act as a templatefor the next circle of replication, achieving exponentiallyamplification of the target DNA. PCR requires a cycle of heating andcooling for denaturation and annealing. Variations of the basic PCRinclude asymmetric PCR [Innis et al., PNAS, vol. 85, 9436-9440 (1988)],inverse PCR [Ochman et al., Genetics, vol. 120(3), 621-623, (1988)],reverse transcription PCR (RT-PCR) (Freeman et al., BioTechniques, vol.26(1), 112-22, 124-5 (1999), the contents of which are incorporatedherein by reference in their entirety and so on). In RT-PCR, a singlestranded RNA is the desired target and is converted to a double strandedDNA first by reverse transcriptase.

A variety of isothermal in vitro nucleic acid amplification techniqueshave been developed as alternatives or complements of PCR. For example,strand displacement amplification (SDA) is based on the ability of arestriction enzyme to form a nick. (Walker et al., PNAS, vol. 89,392-396 (1992), the contents of which are incorporated herein byreference in their entirety)). A restriction enzyme recognition sequenceis inserted into an annealed primer sequence. Primers are extended by aDNA polymerase and dNTPs to form a duplex. Only one strand of the duplexis cleaved by the restriction enzyme. Each single strand chain is thenavailable as a template for subsequent synthesis. SDA does not requirethe complicated temperature control cycle of PCR.

Nucleic acid sequence-based amplification (NASBA), also calledtranscription mediated amplification (TMA), is also an isothermalamplification method that utilizes a combination of DNA polymerase,reverse transcriptase, RNAse H, and T7 RNA polymerase. [Compton, Nature,vol. 350, 91-92 (1991)] the contents of which are incorporated herein byreference in their entirety. A target RNA is used as a template and areverse transcriptase synthesizes its complementary DNA strand. RNAse Hhydrolyzes the RNA template, making space for a DNA polymerase tosynthesize a DNA strand complementary to the first DNA strand which iscomplementary to the RNA target, forming a DNA duplex. T7 RNA polymerasecontinuously generates complementary RNA strands of this DNA duplex.These RNA strands act as templates for new cycles of DNA synthesis,resulting in amplification of the target gene.

Rolling-circle amplification (RCA) amplifies a single stranded circularpolynucleotide and involves numerous rounds of isothermal enzymaticsynthesis where D29 DNA polymerase extends a primer by continuouslyprogressing around the polynucleotide circle to replicate its sequenceover and over again. Therefore, a linear copy of the circular templateis achieved. A primer can then be annealed to this linear copy and itscomplementary chain can be synthesized. [See Lizardi et al., NatureGenetics, vol. 19, 225-232 (1998)] the contents of which areincorporated herein by reference in their entirety. A single strandedcircular DNA can also serve as a template for RNA synthesis in thepresence of an RNA polymerase. (Daubendiek et al., JACS, vol. 117,7818-7819 (1995), the contents of which are incorporated herein byreference in their entirety). An inverse rapid amplification of cDNAends (RACE) RCA is described by Polidoros et al. A messenger RNA (mRNA)is reverse transcribed into cDNA, followed by RNAse H treatment toseparate the cDNA. The cDNA is then circularized by CircLigase into acircular DNA. The amplification of the resulting circular DNA isachieved with RCA. (Polidoros et al., BioTechniques, vol. 41, 35-42(2006), the contents of which are incorporated herein by reference intheir entirety).

Any of the foregoing methods can be utilized in the manufacture of oneor more regions of the polynucleotides of the present disclosure.

Assembling polynucleotides or nucleic acids by a ligase is also widelyused. DNA or RNA ligases promote intermolecular ligation of the 5′ and3′ ends of polynucleotide chains through the formation of aphosphodiester bond. Ligase chain reaction (LCR) is a promisingdiagnosing technique based on the principle that two adjacentpolynucleotide probes hybridize to one strand of a target gene andcouple to each other by a ligase. If a target gene is not present, or ifthere is a mismatch at the target gene, such as a single-nucleotidepolymorphism (SNP), the probes cannot ligase. (Wiedmann et al., PCRMethods and Application, vol. 3 (4), s51-s64 (1994), the contents ofwhich are incorporated herein by reference in their entirety). LCR canbe combined with various amplification techniques to increasesensitivity of detection or to increase the amount of products if it isused in synthesizing polynucleotides and nucleic acids.

Several library preparation kits for nucleic acids are now commerciallyavailable. They include enzymes and buffers to convert a small amount ofnucleic acid samples into an indexed library for downstreamapplications. For example, DNA fragments can be placed in a NEBNEXT®ULTRA™ DNA Library Prep Kit by NEWENGLAND BIOLABS® for end preparation,ligation, size selection, clean-up, PCR amplification and finalclean-up.

Continued development is going on to improvement the amplificationtechniques. For example, U.S. Pat. No. 8,367,328 to Asada et al. thecontents of which are incorporated herein by reference in theirentirety, teaches utilizing a reaction enhancer to increase theefficiency of DNA synthesis reactions by DNA polymerases. The reactionenhancer comprises an acidic substance or cationic complexes of anacidic substance. U.S. Pat. No. 7,384,739 to Kitabayashi et al. thecontents of which are incorporated herein by reference in theirentirety, teaches a carboxylate ion-supplying substance that promotesenzymatic DNA synthesis, wherein the carboxylate ion-supplying substanceis selected from oxalic acid, malonic acid, esters of oxalic acid,esters of malonic acid, salts of malonic acid, and esters of maleicacid. U.S. Pat. No. 7,378,262 to Sobek et al. the contents of which areincorporated herein by reference in their entirety, discloses an enzymecomposition to increase fidelity of DNA amplifications. The compositioncomprises one enzyme with 3′ exonuclease activity but no polymeraseactivity and another enzyme that is a polymerase. Both of the enzymesare thermostable and are reversibly modified to be inactive at lowertemperatures.

U.S. Pat. No. 7,550,264 to Getts et al. teaches multiple round ofsynthesis of sense RNA molecules are performed by attachingoligodeoxynucleotides tails onto the 3′ end of cDNA molecules andinitiating RNA transcription using RNA polymerase, the contents of whichare incorporated herein by reference in their entirety. US Pat.Publication No. 2013/0183718 to Rohayem teaches RNA synthesis byRNA-dependent RNA polymerases (RdRp) displaying an RNA polymeraseactivity on single-stranded DNA templates, the contents of which areincorporated herein by reference in their entirety. Oligonucleotideswith non-standard nucleotides can be synthesized with enzymaticpolymerization by contacting a template comprising non-standardnucleotides with a mixture of nucleotides that are complementary to thenucleotides of the template as disclosed in U.S. Pat. No. 6,617,106 toBenner, the contents of which are incorporated herein by reference intheir entirety.

Chemical Synthesis

Standard methods can be applied to synthesize an isolated polynucleotidesequence encoding an isolated polypeptide of interest. For example, asingle DNA or RNA oligomer containing a codon-optimized nucleotidesequence coding for the particular isolated polypeptide can besynthesized. In other aspects, several small oligonucleotides coding forportions of the desired polypeptide can be synthesized and then ligated.In some aspects, the individual oligonucleotides typically contain 5′ or3′ overhangs for complementary assembly.

A polynucleotide disclosed herein (e.g., mRNA) can be chemicallysynthesized using chemical synthesis methods and potential nucleobasesubstitutions known in the art. See, for example, InternationalPublication Nos. WO2014093924, WO2013052523; WO2013039857, WO2012135805,WO2013151671; U.S. Publ. No. US20130115272; or U.S. Pat. Nos. 8,999,380,8,710,200, all of which are herein incorporated by reference in theirentireties.

V. PURIFICATION AND QUANTITATION OF POLYNUCLEOTIDES Purification

Purification of the polynucleotides described herein (i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide) caninclude, but is not limited to, polynucleotide clean-up, qualityassurance and quality control. Clean-up can be performed by methodsknown in the arts such as, but not limited to, AGENCOURT® beads (BeckmanCoulter Genomics, Danvers, Mass.), poly-T beads, LNA™ oligo-T captureprobes (EXIQON® Inc., Vedbaek, Denmark) or HPLC based purificationmethods such as, but not limited to, strong anion exchange HPLC, weakanion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobicinteraction HPLC (HIC-HPLC). The term “purified” when used in relationto a polynucleotide such as a “purified polynucleotide” refers to onethat is separated from at least one contaminant. As used herein, a“contaminant” is any substance that makes another unfit, impure orinferior. Thus, a purified polynucleotide (e.g., DNA and RNA) is presentin a form or setting different from that in which it is found in nature,or a form or setting different from that which existed prior tosubjecting it to a treatment or purification method.

In some embodiments, purification of a polynucleotide of the disclosureremoves impurities that can reduce or remove an unwanted immuneresponse, e.g., reducing cytokine activity.

In some embodiments, the polynucleotide of the disclosure is purifiedprior to administration using column chromatography (e.g., strong anionexchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC),and hydrophobic interaction HPLC (HIC-HPLC), or (LCMS)). In someembodiments, a column chromatography (e.g., strong anion exchange HPLC,weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobicinteraction HPLC (HIC-HPLC), or (LCMS)) purified polynucleotide, thatencodes an MCM polypeptide disclosed herein increases expression of MCMcompared to polynucleotides encoding MCM purified by a differentpurification method. In some embodiments, a column chromatography (e.g.,strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), or (LCMS))purified polynucleotide encodes an MCM polypeptide or functionalfragment thereof comprising one or more of the point mutations V69,T499, H532, A598, and V671. In some embodiments, the RP-HPLC purifiedpolynucleotide increases MCM expression (e.g., by 20-50%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, orat least 50%).

A quality assurance and/or quality control check can be conducted usingmethods such as, but not limited to, gel electrophoresis, UV absorbance,or analytical HPLC.

In another embodiment, the polynucleotides can be sequenced by methodsincluding, but not limited to reverse-transcriptase-PCR.

Quantification

In some embodiments, the polynucleotides of the present disclosure(i.e., a polynucleotide comprising an ORF encoding an MCM polypeptide)can be quantified in exosomes or when derived from one or more bodilyfluid. As used herein “bodily fluids” include peripheral blood, serum,plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bonemarrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breastmilk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper'sfluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cystfluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme,chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginalsecretions, mucosal secretion, stool water, pancreatic juice, lavagefluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavityfluid, and umbilical cord blood. Alternatively, exosomes can beretrieved from an organ selected from the group consisting of lung,heart, pancreas, stomach, intestine, bladder, kidney, ovary, testis,skin, colon, breast, prostate, brain, esophagus, liver, and placenta.

In the exosome quantification method, a sample of not more than 2 mL isobtained from the subject and the exosomes isolated by size exclusionchromatography, density gradient centrifugation, differentialcentrifugation, nanomembrane ultrafiltration, immunoabsorbent capture,affinity purification, microfluidic separation, or combinations thereof.In the analysis, the level or concentration of a polynucleotide can bean expression level, presence, absence, truncation or alteration of theadministered construct. It is advantageous to correlate the level withone or more clinical phenotypes or with an assay for a human diseasebiomarker. The assay can be performed using construct specific probes,cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis,mass spectrometry, or combinations thereof while the exosomes can beisolated using immunohistochemical methods such as enzyme linkedimmunosorbent assay (ELISA) methods. Exosomes can also be isolated bysize exclusion chromatography, density gradient centrifugation,differential centrifugation, nanomembrane ultrafiltration,immunoabsorbent capture, affinity purification, microfluidic separation,or combinations thereof.

These methods afford the investigator the ability to monitor, in realtime, the level of polynucleotides remaining or delivered. This ispossible because the polynucleotides of the present disclosure differfrom the endogenous forms due to the structural or chemicalmodifications.

In some embodiments, the polynucleotide can be quantified using methodssuch as, but not limited to, ultraviolet visible spectroscopy (UV/Vis).A non-limiting example of a UV/Vis spectrometer is a NANODROP®spectrometer (ThermoFisher, Waltham, Mass.). The quantifiedpolynucleotide can be analyzed in order to determine if thepolynucleotide can be of proper size, check that no degradation of thepolynucleotide has occurred. Degradation of the polynucleotide can bechecked by methods such as, but not limited to, agarose gelelectrophoresis, HPLC based purification methods such as, but notlimited to, strong anion exchange HPLC, weak anion exchange HPLC,reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC(HIC-HPLC), liquid chromatography-mass spectrometry (LCMS), capillaryelectrophoresis (CE) and capillary gel electrophoresis (CGE).

VI. PHARMACEUTICAL COMPOSITIONS

The disclosure includes pharmaceutical compositions that comprise thepolynucleotide described herein, i.e., a polynucleotide comprising anORF encoding an MCM polypeptide. In some embodiments, the formulationcan contain polynucleotide encoding wild type MCM or MCM comprising anucleotide sequence having significant sequence similarity to apolynucleotide selected from the group of SEQ ID NOs: 1-207, 732-765,and 772, wherein the ORF encodes an MCM polypeptide.

Pharmaceutical compositions can optionally comprise one or moreadditional active substances, e.g., therapeutically and/orprophylactically active substances. Pharmaceutical compositions of thepresent disclosure can be sterile and/or pyrogen-free. Generalconsiderations in the formulation and/or manufacture of pharmaceuticalagents can be found, for example, in Remington: The Science and Practiceof Pharmacy 21^(st) ed., Lippincott Williams & Wilkins, 2005(incorporated herein by reference in its entirety). For the purposes ofthe present disclosure, the phrase “active ingredient” generally refersto polynucleotides to be delivered as described herein.

Formulations of the pharmaceutical compositions described herein can beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofassociating the active ingredient with an excipient and/or one or moreother accessory ingredients.

A pharmaceutical composition in accordance with the present disclosurecan be prepared, packaged, and/or sold in bulk, as a single unit dose,and/or as a plurality of single unit doses. As used herein, a “unitdose” refers to a discrete amount of the pharmaceutical compositioncomprising a predetermined amount of the active ingredient. The amountof the active ingredient is generally equal to the dosage of the activeingredient that would be administered to a subject and/or a convenientfraction of such a dosage such as, for example, one-half or one-third ofsuch a dosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the present disclosure canvary, depending upon the identity, size, and/or condition of the subjectbeing treated and further depending upon the route by which thecomposition is to be administered. For example, the composition cancomprise between 0.1% and 99% (w/w) of the active ingredient. By way ofexample, the composition can comprise between 0.1% and 100%, e.g.,between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w)active ingredient.

In some embodiments, the formulations described herein can contain atleast one polynucleotide. As a non-limiting example, the formulationscan contain 1, 2, 3, 4 or 5 polynucleotides.

In some embodiments, the formulations described herein can comprise morethan one type of polynucleotide. In some embodiments, the formulationcan comprise a polynucleotide in linear and circular form. In anotherembodiment, the formulation can comprise a circular polynucleotide andan IVT polynucleotide. In yet another embodiment, the formulation cancomprise an IVT polynucleotide, a chimeric polynucleotide and a circularpolynucleotide.

In some embodiments, compositions are administered to humans, humanpatients or subjects. Although the descriptions of pharmaceuticalcompositions provided herein are principally directed to pharmaceuticalcompositions that are suitable for administration to humans, it will beunderstood by the skilled artisan that such compositions are generallysuitable for administration to any other animal, e.g., to non-humananimals, e.g. non-human mammals. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and/or perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions is contemplated include, but are not limited to, humansand/or other primates; mammals, including commercially relevant mammalssuch as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats;and/or birds, including commercially relevant birds such as poultry,chickens, ducks, geese, and/or turkeys.

Delivery Agents

a. Lipid Compound

The present disclosure provides pharmaceutical compositions withadvantageous properties. In particular, the present application providespharmaceutical compositions comprising:

(a) a polynucleotide comprising an ORF encoding an MCM polypeptide; and

(b) a lipid compound having the formula (I)

wherein

R₁ is selected from the group consisting of C₅₋₂₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a carbocycle, heterocycle, —OR,—O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —N(R)₂,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, and—C(R)N(R)₂C(O)OR, and each n is independently selected from 1, 2, 3, 4,and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, orsalts or stereoisomers thereof, wherein alkyl and alkenyl groups can belinear or branched.

In some embodiments, a subset of compounds of Formula (I) includes thosein which when R₄ is —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, or —CQ(R)₂, then(i) Q is not —N(R)₂ when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or7-membered heterocycloalkyl when n is 1 or 2.

In another embodiments, another subset of compounds of Formula (I)includes those in which

R₁ is selected from the group consisting of C₅₋₂₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheteroaryl having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, and a 5- to 14-membered heterocycloalkyl having one ormore heteroatoms selected from N, O, and S which is substituted with oneor more substituents selected from oxo (═O), OH, amino, and C₁₋₃ alkyl,and each n is independently selected from 1, 2, 3, 4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or stereoisomers thereof.

In yet another embodiments, another subset of compounds of Formula (I)includes those in which

R₁ is selected from the group consisting of C₅₋₂₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheterocycle having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, and each n is independently selected from 1, 2, 3, 4,and 5; and when Q is a 5- to 14-membered heterocycle and (i) R₄ is—(CH₂)_(n)Q in which n is 1 or 2, or (ii) R₄ is —(CH₂)_(n)CHQR in whichn is 1, or (iii) R₄ is —CHQR, and —CQ(R)₂, then Q is either a 5- to14-membered heteroaryl or 8- to 14-membered heterocycloalkyl;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or stereoisomers thereof.

In still another embodiments, another subset of compounds of Formula (I)includes those in which

R₁ is selected from the group consisting of C₅₋₂₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheteroaryl having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, and each n is independently selected from 1, 2, 3, 4,and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or stereoisomers thereof.

In yet another embodiments, another subset of compounds of Formula (I)includes those in which

R₁ is selected from the group consisting of C₅₋₂₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₂₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is —(CH₂)_(n)Q or —(CH₂)_(n)CHQR, where Q is —N(R)₂, and n isselected from 3, 4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₁₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or stereoisomers thereof.

In still another embodiments, another subset of compounds of Formula (I)includes those in which

R₁ is selected from the group consisting of C₅₋₂₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of C₁₋₁₄alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃, togetherwith the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of —(CH₂)_(n)Q, —(CH₂)_(n)CHQR,—CHQR, and —CQ(R)₂, where Q is —N(R)₂, and n is selected from 1, 2, 3,4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₁₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or stereoisomers thereof.

In certain embodiments, a subset of compounds of Formula (I) includesthose of Formula (IA):

or a salt or stereoisomer thereof, wherein 1 is selected from 1, 2, 3,4, and 5; m is selected from 5, 6, 7, 8, and 9; M₁ is a bond or M′; R₄is unsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which Q is OH,—NHC(S)N(R)₂, or —NHC(O)N(R)₂; M and M′ are independently selected from—C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, an aryl group, and aheteroaryl group; and

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

In certain embodiments, a subset of compounds of Formula (I) includesthose of Formula (II):

or a salt or stereoisomer thereof, wherein 1 is selected from 1, 2, 3,4, and 5; M₁ is a bond or M′; R₄ is unsubstituted C₁₋₃ alkyl, or—(CH₂)_(n)Q, in which n is 2, 3, or 4, and Q is OH, —NHC(S)N(R)₂, or—NHC(O)N(R)₂; M and M′ are independently selected from —C(O)O—, —OC(O)—,—C(O)N(R′)—, —P(O)(OR′)O—, an aryl group, and a heteroaryl group; and

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

In some embodiments, the compound of formula (I) is of the formula(IIa),

or a salt thereof, wherein R₄ is as described above.

In some embodiments, the compound of formula (I) is of the formula(IIb),

or a salt thereof, wherein R₄ is as described above.

In some embodiments, the compound of formula (I) is of the formula(IIc),

or a salt thereof, wherein R₄ is as described above.

In some embodiments, the compound of formula (I) is of the formula(IIe):

or a salt thereof, wherein R₄ is as described above.

In some embodiments, the compound of formula (IIa), (IIb), (IIc), or(IIe) comprises an R₄ which is selected from —(CH₂)_(n)Q and—(CH₂)_(n)CHQR, wherein Q, R and n are as defined above.

In some embodiments, Q is selected from the group consisting of —OR,—OH, —O(CH₂)_(n)N(R)₂, —OC(O)R, —CX₃, —CN, —N(R)C(O)R, —N(H)C(O)R,—N(R)S(O)₂R, —N(H)S(O)₂R, —N(R)C(O)N(R)₂, —N(H)C(O)N(R)₂,—N(H)C(O)N(H)(R), —N(R)C(S)N(R)₂, —N(H)C(S)N(R)₂, —N(H)C(S)N(H)(R), anda heterocycle, wherein R is as defined above. In some aspects, n is 1 or2. In some embodiments, Q is OH, —NHC(S)N(R)₂, or —NHC(O)N(R)₂.

In some embodiments, the compound of formula (I) is of the formula(IId),

or a salt thereof, wherein R₂ and R₃ are independently selected from thegroup consisting of C₅₋₁₄ alkyl and C₅₋₁₄ alkenyl, n is selected from 2,3, and 4, and R′, R″, R₅, R₆ and m are as defined above.

In some aspects of the compound of formula (IId), R₂ is C₈ alkyl. Insome aspects of the compound of formula (IId), R₃ is C₅-C₉ alkyl. Insome aspects of the compound of formula (IId), m is 5, 7, or 9. In someaspects of the compound of formula (IId), each R₅ is H. In some aspectsof the compound of formula (IId), each R₆ is H.

In another aspect, the present application provides a lipid composition(e.g., a lipid nanoparticle (LNP)) comprising: (1) a compound having theformula (I); (2) optionally a helper lipid (e.g. a phospholipid); (3)optionally a structural lipid (e.g. a sterol); (4) optionally a lipidconjugate (e.g. a PEG-lipid); and (5) optionally a quaternary aminecompound. In exemplary embodiments, the lipid composition (e.g., LNP)further comprises a polynucleotide encoding an MCM polypeptide, e.g., apolynucleotide encapsulated therein.

As used herein, the term “alkyl” or “alkyl group” means a linear orbranched, saturated hydrocarbon including one or more carbon atoms(e.g., one, two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen,eighteen, nineteen, twenty, or more carbon atoms).

The notation “C₁₋₁₄ alkyl” means a linear or branched, saturatedhydrocarbon including 1-14 carbon atoms. An alkyl group can beoptionally substituted.

As used herein, the term “alkenyl” or “alkenyl group” means a linear orbranched hydrocarbon including two or more carbon atoms (e.g., two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen,twenty, or more carbon atoms) and at least one double bond.

The notation “C₂₋₁₄ alkenyl” means a linear or branched hydrocarbonincluding 2-14 carbon atoms and at least one double bond. An alkenylgroup can include one, two, three, four, or more double bonds. Forexample, Cis alkenyl can include one or more double bonds. A Cis alkenylgroup including two double bonds can be a linoleyl group. An alkenylgroup can be optionally substituted.

As used herein, the term “carbocycle” or “carbocyclic group” means amono- or multi-cyclic system including one or more rings of carbonatoms. Rings can be three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, or fifteen membered rings.

The notation “C₃₋₆ carbocycle” means a carbocycle including a singlering having 3-6 carbon atoms. Carbocycles can include one or more doublebonds and can be aromatic (e.g., aryl groups). Examples of carbocyclesinclude cyclopropyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, and1,2-dihydronaphthyl groups. Carbocycles can be optionally substituted.

As used herein, the term “heterocycle” or “heterocyclic group” means amono- or multi-cyclic system including one or more rings, where at leastone ring includes at least one heteroatom. Heteroatoms can be, forexample, nitrogen, oxygen, or sulfur atoms. Rings can be three, four,five, six, seven, eight, nine, ten, eleven, or twelve membered rings.Heterocycles can include one or more double bonds and can be aromatic(e.g., heteroaryl groups). Examples of heterocycles include imidazolyl,imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl,pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl,isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furyl,tetrahydrofuryl, thiophenyl, pyridinyl, piperidinyl, quinolyl, andisoquinolyl groups. Heterocycles can be optionally substituted.

As used herein, a “biodegradable group” is a group that can facilitatefaster metabolism of a lipid in a subject. A biodegradable group can be,but is not limited to, —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—,—C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, anaryl group, and a heteroaryl group.

As used herein, an “aryl group” is a carbocyclic group including one ormore aromatic rings. Examples of aryl groups include phenyl and naphthylgroups.

As used herein, a “heteroaryl group” is a heterocyclic group includingone or more aromatic rings. Examples of heteroaryl groups includepyrrolyl, furyl, thiophenyl, imidazolyl, oxazolyl, and thiazolyl. Botharyl and heteroaryl groups can be optionally substituted. For example, Mand M′ can be selected from the non-limiting group consisting ofoptionally substituted phenyl, oxazole, and thiazole. In the formulasherein, M and M′ can be independently selected from the list ofbiodegradable groups above.

Alkyl, alkenyl, and cyclyl (e.g., carbocyclyl and heterocyclyl) groupscan be optionally substituted unless otherwise specified. Optionalsubstituents can be selected from the group consisting of, but are notlimited to, a halogen atom (e.g., a chloride, bromide, fluoride, oriodide group), a carboxylic acid (e.g., —C(O)OH), an alcohol (e.g., ahydroxyl, —OH), an ester (e.g., —C(O)OR or —OC(O)R), an aldehyde (e.g.,—C(O)H), a carbonyl (e.g., —C(O)R, alternatively represented by C═O), anacyl halide (e.g., —C(O)X, in which X is a halide selected from bromide,fluoride, chloride, and iodide), a carbonate (e.g., —OC(O)OR), an alkoxy(e.g., —OR), an acetal (e.g., —C(OR)₂R″″, in which each OR are alkoxygroups that can be the same or different and R″″ is an alkyl or alkenylgroup), a phosphate (e.g., P(O)₄ ³⁻), a thiol (e.g., —SH), a sulfoxide(e.g., —S(O)R), a sulfinic acid (e.g., —S(O)OH), a sulfonic acid (e.g.,—S(O)₂OH), a thial (e.g., —C(S)H), a sulfate (e.g., S(O)₄ ²⁻), asulfonyl (e.g., —S(O)₂—), an amide (e.g., —C(O)NR₂, or —N(R)C(O)R), anazido (e.g., —N₃), a nitro (e.g., —NO₂), a cyano (e.g., —CN), anisocyano (e.g., —NC), an acyloxy (e.g., —OC(O)R), an amino (e.g., —NR₂,—NRH, or —NH₂), a carbamoyl (e.g., —OC(O)NR₂, —OC(O)NRH, or —OC(O)NH₂),a sulfonamide (e.g., —S(O)₂NR₂, —S(O)₂NRH, —S(O)₂NH₂, —N(R)S(O)₂R,—N(H)S(O)₂R, —N(R)S(O)₂H, or —N(H)S(O)₂H), an alkyl group, an alkenylgroup, and a cyclyl (e.g., carbocyclyl or heterocyclyl) group.

In any of the preceding, R is an alkyl or alkenyl group, as definedherein. In some embodiments, the substituent groups themselves can befurther substituted with, for example, one, two, three, four, five, orsix substituents as defined herein. For example, a C₁₋₆ alkyl group canbe further substituted with one, two, three, four, five, or sixsubstituents as described herein.

The compounds of any one of formulae (I), (IA), (II), (IIa), (IIb),(IIc), (IId), and (IIe) include one or more of the following featureswhen applicable.

In some embodiments, R₄ is selected from the group consisting of a C₃₋₆carbocycle, —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, and —CQ(R)₂, where Q isselected from a C₃₋₆ carbocycle, 5- to 14-membered aromatic ornon-aromatic heterocycle having one or more heteroatoms selected from N,O, S, and P, —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H,—CXH₂, —CN, —N(R)₂, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂,—N(R)C(S)N(R)₂, and —C(R)N(R)₂C(O)OR, and each n is independentlyselected from 1, 2, 3, 4, and 5.

In another embodiment, R₄ is selected from the group consisting of aC₃₋₆ carbocycle, —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, and —CQ(R)₂, whereQ is selected from a C₃₋₆ carbocycle, a 5- to 14-membered heteroarylhaving one or more heteroatoms selected from N, O, and S, —OR,—O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —C(O)N(R)₂,—N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—C(R)N(R)₂C(O)OR, and a 5- to 14-membered heterocycloalkyl having one ormore heteroatoms selected from N, O, and S which is substituted with oneor more substituents selected from oxo (═O), OH, amino, and C₁₋₃ alkyl,and each n is independently selected from 1, 2, 3, 4, and 5.

In another embodiment, R₄ is selected from the group consisting of aC₃₋₆ carbocycle, —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, and —CQ(R)₂, whereQ is selected from a C₃₋₆ carbocycle, a 5- to 14-membered heterocyclehaving one or more heteroatoms selected from N, O, and S, —OR,—O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —C(O)N(R)₂,—N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—C(R)N(R)₂C(O)OR, and each n is independently selected from 1, 2, 3, 4,and 5; and when Q is a 5- to 14-membered heterocycle and (i) R₄ is—(CH₂)_(n)Q in which n is 1 or 2, or (ii) R₄ is —(CH₂)_(n)CHQR in whichn is 1, or (iii) R₄ is —CHQR, and —CQ(R)₂, then Q is either a 5- to14-membered heteroaryl or 8- to 14-membered heterocycloalkyl.

In another embodiment, R₄ is selected from the group consisting of aC₃₋₆ carbocycle, —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, and —CQ(R)₂, whereQ is selected from a C₃₋₆ carbocycle, a 5- to 14-membered heteroarylhaving one or more heteroatoms selected from N, O, and S, —OR,—O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —C(O)N(R)₂,—N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—C(R)N(R)₂C(O)OR, and each n is independently selected from 1, 2, 3, 4,and 5.

In another embodiment, R₄ is unsubstituted C₁₋₄ alkyl, e.g.,unsubstituted methyl.

In certain embodiments, the disclosure provides a compound having theFormula (I), wherein R₄ is —(CH₂)_(n)Q or —(CH₂)_(n)CHQR, where Q is—N(R)₂, and n is selected from 3, 4, and 5.

In certain embodiments, the disclosure provides a compound having theFormula (I), wherein R₄ is selected from the group consisting of—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, and —CQ(R)₂, where Q is —N(R)₂, andn is selected from 1, 2, 3, 4, and 5.

In certain embodiments, the disclosure provides a compound having theFormula (I), wherein R₂ and R₃ are independently selected from the groupconsisting of C₂₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, orR₂ and R₃, together with the atom to which they are attached, form aheterocycle or carbocycle, and R₄ is —(CH₂)_(n)Q or —(CH₂)_(n)CHQR,where Q is —N(R)₂, and n is selected from 3, 4, and 5.

In certain embodiments, R₂ and R₃ are independently selected from thegroup consisting of C₂₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and—R*OR″, or R₂ and R₃, together with the atom to which they are attached,form a heterocycle or carbocycle.

In some embodiments, R₁ is selected from the group consisting of C₅₋₂₀alkyl and C₅₋₂₀ alkenyl.

In other embodiments, R₁ is selected from the group consisting of—R*YR″, —YR″, and —R″M′R′.

In certain embodiments, R₁ is selected from —R*YR″ and —YR″. In someembodiments, Y is a cyclopropyl group. In some embodiments, R* is C₈alkyl or C₈ alkenyl. In certain embodiments, R″ is C₃₋₁₂ alkyl. Forexample, R″ can be C₃ alkyl. For example, R″ can be C₄₋₈ alkyl (e.g.,C₄, C₅, C₆, C₇, or C₈ alkyl).

In some embodiments, R₁ is C₅₋₂₀ alkyl. In some embodiments, R₁ is C₆alkyl. In some embodiments, R₁ is C₈ alkyl. In other embodiments, R₁ isC₉ alkyl. In certain embodiments, R₁ is C₁₄ alkyl. In other embodiments,R₁ is C₁₈ alkyl.

In some embodiments, R₁ is C₅₋₂₀ alkenyl. In certain embodiments, R₁ isC₁₈ alkenyl. In some embodiments, R₁ is linoleyl.

In certain embodiments, R₁ is branched (e.g., decan-2-yl, undecan-3-yl,dodecan-4-yl, tridecan-5-yl, tetradecan-6-yl, 2-methylundecan-3-yl,2-methyldecan-2-yl, 3-methylundecan-3-yl, 4-methyldodecan-4-yl, orheptadeca-9-yl). In certain embodiments, R₁ is

In certain embodiments, R₁ is unsubstituted C₅₋₂₀ alkyl or C₅₋₂₀alkenyl. In certain embodiments, R′ is substituted C₅₋₂₀ alkyl or C₅₋₂₀alkenyl (e.g., substituted with a C₃₋₆ carbocycle such as1-cyclopropylnonyl).

In other embodiments, R₁ is —R″M′R′.

In some embodiments, R′ is selected from —R*YR″ and —YR″. In someembodiments, Y is C₃₋₈ cycloalkyl. In some embodiments, Y is C₆₋₁₀ aryl.In some embodiments, Y is a cyclopropyl group. In some embodiments, Y isa cyclohexyl group. In certain embodiments, R* is C₁ alkyl.

In some embodiments, R″ is selected from the group consisting of C₃₋₁₂alkyl and C₃₋₁₂ alkenyl. In some embodiments, R″ adjacent to Y is C₁alkyl. In some embodiments, R″ adjacent to Y is C₄₋₉ alkyl (e.g., C₄,C₅, C₆, C₇ or C₈ or C₉ alkyl).

In some embodiments, R′ is selected from C₄ alkyl and C₄ alkenyl. Incertain embodiments, R′ is selected from C₅ alkyl and C₅ alkenyl. Insome embodiments, R′ is selected from C₆ alkyl and C₆ alkenyl. In someembodiments, R′ is selected from C₇ alkyl and C₇ alkenyl. In someembodiments, R′ is selected from C₉ alkyl and C₉ alkenyl.

In other embodiments, R′ is selected from C₁₁ alkyl and C₁₁ alkenyl. Inother embodiments, R′ is selected from C₁₂ alkyl, C₁₂ alkenyl, C₁₃alkyl, C₁₃ alkenyl, C₁₄ alkyl, C₁₄ alkenyl, C₁₅ alkyl, C₁₅ alkenyl, C₁₆alkyl, C₁₆ alkenyl, C₁₇ alkyl, C₁₇ alkenyl, C₁₈ alkyl, and C₁₈ alkenyl.In certain embodiments, R′ is branched (e.g., decan-2-yl, undecan-3-yl,dodecan-4-yl, tridecan-5-yl, tetradecan-6-yl, 2-methylundecan-3-yl,2-methyldecan-2-yl, 3-methylundecan-3-yl, 4-methyldodecan-4-yl orheptadeca-9-yl). In certain embodiments, R′ is

In certain embodiments, R′ is unsubstituted C-18 alkyl. In certainembodiments, R′ is substituted C₁₋₁₈ alkyl (e.g., C₁₋₁₅ alkylsubstituted with a C₃₋₆ carbocycle such as 1-cyclopropylnonyl).

In some embodiments, R″ is selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl. In some embodiments, R″ is C₃ alkyl, C₄ alkyl,C₅ alkyl, C₆ alkyl, C₇ alkyl, or C₈ alkyl. In some embodiments, R″ is C₉alkyl, C₁₀ alkyl, C₁₁ alkyl, C₁₂ alkyl, C₁₃ alkyl, or C₁₄ alkyl.

In some embodiments, M′ is —C(O)O—. In some embodiments, M′ is —OC(O)—.

In other embodiments, M′ is an aryl group or heteroaryl group. Forexample, M′ can be selected from the group consisting of phenyl,oxazole, and thiazole.

In some embodiments, M is —C(O)O—. In some embodiments, M is —OC(O)—. Insome embodiments, M is —C(O)N(R′)—. In some embodiments, M is—P(O)(OR′)O—.

In other embodiments, M is an aryl group or heteroaryl group. Forexample, M can be selected from the group consisting of phenyl, oxazole,and thiazole.

In some embodiments, M is the same as M′. In other embodiments, M isdifferent from M′.

In some embodiments, each R₅ is H. In certain such embodiments, each R₆is also H.

In some embodiments, R₇ is H. In other embodiments, R₇ is C₁₋₃ alkyl(e.g., methyl, ethyl, propyl, or i-propyl).

In some embodiments, R₂ and R₃ are independently C₅₋₁₄ alkyl or C₅₋₁₄alkenyl.

In some embodiments, R₂ and R₃ are the same. In some embodiments, R₂ andR₃ are C₈ alkyl. In certain embodiments, R₂ and R₃ are C₂ alkyl. Inother embodiments, R₂ and R₃ are C₃ alkyl. In some embodiments, R₂ andR₃ are C₄ alkyl. In certain embodiments, R₂ and R₃ are C₅ alkyl. Inother embodiments, R₂ and R₃ are C₆ alkyl. In some embodiments, R₂ andR₃ are C₇ alkyl.

In other embodiments, R₂ and R₃ are different. In certain embodiments,R₂ is C₈ alkyl. In some embodiments, R₃ is C₁₋₇ (e.g., C₁, C₂, C₃, C₄,C₅, C₆, or C₇ alkyl) or C₉ alkyl.

In some embodiments, R₇ and R₃ are H.

In certain embodiments, R₂ is H.

In some embodiments, m is 5, 7, or 9.

In some embodiments, R₄ is selected from —(CH₂)_(n)Q and —(CH₂)_(n)CHQR.

In some embodiments, Q is selected from the group consisting of —OR,—OH, —O(CH₂)_(n)N(R)₂, —OC(O)R, —CX₃, —CN, —N(R)C(O)R, —N(H)C(O)R,—N(R)S(O)₂R, —N(H)S(O)₂R, —N(R)C(O)N(R)₂, —N(H)C(O)N(R)₂,—N(H)C(O)N(H)(R), —N(R)C(S)N(R)₂, —N(H)C(S)N(R)₂, —N(H)C(S)N(H)(R),—C(R)N(R)₂C(O)OR, a carbocycle, and a heterocycle.

In certain embodiments, Q is —OH.

In certain embodiments, Q is a substituted or unsubstituted 5- to10-membered heteroaryl, e.g., Q is an imidazole, a pyrimidine, a purine,2-amino-1,9-dihydro-6H-purin-6-one-9-yl (or guanin-9-yl), adenin-9-yl,cytosin-1-yl, or uracil-1-yl. In certain embodiments, Q is a substituted5- to 14-membered heterocycloalkyl, e.g., substituted with one or moresubstituents selected from oxo (═O), OH, amino, and C₁₋₃ alkyl. Forexample, Q is 4-methylpiperazinyl, 4-(4-methoxybenzyl)piperazinyl, orisoindolin-2-yl-1,3-dione.

In certain embodiments, Q is an unsubstituted or substituted C₆₋₁₀ aryl(such as phenyl) or C₃₋₆ cycloalkyl.

In some embodiments, n is 1. In other embodiments, n is 2. In furtherembodiments, n is 3. In certain other embodiments, n is 4. For example,R₄ can be —(CH₂)₂OH. For example, R₄ can be —(CH₂)₃OH. For example, R₄can be —(CH₂)₄OH. For example, R₄ can be benzyl. For example, R₄ can be4-methoxybenzyl.

In some embodiments, R₄ is a C₃₋₆ carbocycle. In some embodiments, R₄ isa C₃₋₆ cycloalkyl. For example, R₄ can be cyclohexyl optionallysubstituted with e.g., OH, halo, C₁₋₆ alkyl, etc. For example, R₄ can be2-hydroxycyclohexyl.

In some embodiments, R is H.

In some embodiments, R is unsubstituted C₁₋₃ alkyl or unsubstituted C₂₋₃alkenyl. For example, R₄ can be ˜CH₂CH(OH)CH₃ or ˜CH₂CH(OH)CH₂CH₃.

In some embodiments, R is substituted C₁₋₃ alkyl, e.g., CH₂OH. Forexample, R₄ can be ˜CH₂CH(OH)CH₂OH.

In some embodiments, R₂ and R₃, together with the atom to which they areattached, form a heterocycle or carbocycle. In some embodiments, R₂ andR₃, together with the atom to which they are attached, form a 5- to14-membered aromatic or non-aromatic heterocycle having one or moreheteroatoms selected from N, O, S, and P. In some embodiments, R₂ andR₃, together with the atom to which they are attached, form anoptionally substituted C₃₋₂₀ carbocycle (e.g., C₃₋₁₈ carbocycle, C₃₋₁₅carbocycle, C₃₋₁₂ carbocycle, or C₃₋₁₀ carbocycle), either aromatic ornon-aromatic. In some embodiments, R₂ and R₃, together with the atom towhich they are attached, form a C₃₋₆ carbocycle. In other embodiments,R₂ and R₃, together with the atom to which they are attached, form a C₆carbocycle, such as a cyclohexyl or phenyl group. In certainembodiments, the heterocycle or C₃₋₆ carbocycle is substituted with oneor more alkyl groups (e.g., at the same ring atom or at adjacent ornon-adjacent ring atoms). For example, R₂ and R₃, together with the atomto which they are attached, can form a cyclohexyl or phenyl groupbearing one or more C₅ alkyl substitutions. In certain embodiments, theheterocycle or C₃₋₆ carbocycle formed by R₂ and R₃, is substituted witha carbocycle groups. For example, R₂ and R₃, together with the atom towhich they are attached, can form a cyclohexyl or phenyl group that issubstituted with cyclohexyl. In some embodiments, R₂ and R₃, togetherwith the atom to which they are attached, form a C₇₋₁₅ carbocycle, suchas a cycloheptyl, cyclopentadecanyl, or naphthyl group.

In some embodiments, R₄ is selected from —(CH₂)_(n)Q and —(CH₂)_(n)CHQR.In some embodiments, Q is selected from the group consisting of —OR,—OH, —O(CH₂)_(n)N(R)₂, —OC(O)R, —CX₃, —CN, —N(R)C(O)R, —N(H)C(O)R,—N(R)S(O)₂R, —N(H)S(O)₂R, —N(R)C(O)N(R)₂, —N(H)C(O)N(R)₂,—N(H)C(O)N(H)(R), —N(R)C(S)N(R)₂, —N(H)C(S)N(R)₂, —N(H)C(S)N(H)(R), anda heterocycle. In other embodiments, Q is selected from the groupconsisting of an imidazole, a pyrimidine, and a purine.

In some embodiments, R₂ and R₃, together with the atom to which they areattached, form a heterocycle or carbocycle. In some embodiments, R₂ andR₃, together with the atom to which they are attached, form a C₃₋₆carbocycle, such as a phenyl group. In certain embodiments, theheterocycle or C₃₋₆ carbocycle is substituted with one or more alkylgroups (e.g., at the same ring atom or at adjacent or non-adjacent ringatoms). For example, R₂ and R₃, together with the atom to which they areattached, can form a phenyl group bearing one or more C₅ alkylsubstitutions.

In some embodiments, the pharmaceutical compositions of the presentdisclosure, the compound of formula (I) is selected from the groupconsisting of:

and salts or stereoisomers thereof.

The central amine moiety of a lipid according to formula (I) istypically protonated (i.e., positively charged) at a pH below the pKa ofthe amino moiety and is substantially not charged at a pH above the pKa.Such lipids can be referred to ionizable amino lipids.

In one specific embodiment, the compound of formula (I) is Compound 18.

In some embodiments, the amount of the compound of formula (I) rangesfrom about 1 mol % to 99 mol % in the lipid composition.

In one embodiment, the amount of the compound of formula (I) is at leastabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, or 99 mol % in the lipid composition.

In one embodiment, the amount of the compound of formula (I) ranges fromabout 30 mol % to about 70 mol %, from about 35 mol % to about 65 mol %,from about 40 mol % to about 60 mol %, and from about 45 mol % to about55 mol % in the lipid composition.

In one specific embodiment, the amount of the compound of formula (I) isabout 50 mol % in the lipid composition.

In addition to the compound of formula (I), the lipid composition of thepharmaceutical compositions disclosed herein can comprise additionalcomponents such as phospholipids, structural lipids, quaternary aminecompounds, PEG-lipids, and any combination thereof.

b. Additional Components in the Lipid Composition

(i) Phospholipids

The lipid composition of the pharmaceutical composition disclosed hereincan comprise one or more phospholipids, for example, one or moresaturated or (poly)unsaturated phospholipids or a combination thereof.In general, phospholipids comprise a phospholipid moiety and one or morefatty acid moieties. For example, a phospholipid can be a lipidaccording to formula (III):

in which R_(p) represents a phospholipid moiety and R₁ and R₂ representfatty acid moieties with or without unsaturation that can be the same ordifferent.

A phospholipid moiety can be selected, for example, from thenon-limiting group consisting of phosphatidyl choline, phosphatidylethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidicacid, 2-lysophosphatidyl choline, and a sphingomyelin.

A fatty acid moiety can be selected, for example, from the non-limitinggroup consisting of lauric acid, myristic acid, myristoleic acid,palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleicacid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid,arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoicacid, and docosahexaenoic acid.

Particular phospholipids can facilitate fusion to a membrane. Forexample, a cationic phospholipid can interact with one or morenegatively charged phospholipids of a membrane (e.g., a cellular orintracellular membrane). Fusion of a phospholipid to a membrane canallow one or more elements (e.g., a therapeutic agent) of alipid-containing composition (e.g., LNPs) to pass through the membranepermitting, e.g., delivery of the one or more elements to a targettissue (e.g., tumoral tissue).

Non-natural phospholipid species including natural species withmodifications and substitutions including branching, oxidation,cyclization, and alkynes are also contemplated. For example, aphospholipid can be functionalized with or cross-linked to one or morealkynes (e.g., an alkenyl group in which one or more double bonds isreplaced with a triple bond). Under appropriate reaction conditions, analkyne group can undergo a copper-catalyzed cycloaddition upon exposureto an azide. Such reactions can be useful in functionalizing a lipidbilayer of a nanoparticle composition to facilitate membrane permeationor cellular recognition or in conjugating a nanoparticle composition toa useful component such as a targeting or imaging moiety (e.g., a dye).

Phospholipids include, but are not limited to, glycerophospholipids suchas phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines,phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids.Phospholipids also include phosphosphingolipid, such as sphingomyelin.In some embodiments, a pharmaceutical composition for intratumoraldelivery disclosed herein can comprise more than one phospholipid. Whenmore than one phospholipid is used, such phospholipids can belong to thesame phospholipid class (e.g., MSPC and DSPC) or different classes(e.g., MSPC and MSPE).

Phospholipids can be of a symmetric or an asymmetric type. As usedherein, the term “symmetric phospholipid” includes glycerophospholipidshaving matching fatty acid moieties and sphingolipids in which thevariable fatty acid moiety and the hydrocarbon chain of the sphingosinebackbone include a comparable number of carbon atoms. As used herein,the term “asymmetric phospholipid” includes lysolipids,glycerophospholipids having different fatty acid moieties (e.g., fattyacid moieties with different numbers of carbon atoms and/orunsaturations (e.g., double bonds)), and sphingolipids in which thevariable fatty acid moiety and the hydrocarbon chain of the sphingosinebackbone include a dissimilar number of carbon atoms (e.g., the variablefatty acid moiety include at least two more carbon atoms than thehydrocarbon chain or at least two fewer carbon atoms than thehydrocarbon chain).

In some embodiments, the lipid composition of a pharmaceuticalcomposition disclosed herein comprises at least one symmetricphospholipid. Symmetric phospholipids can be selected from thenon-limiting group consisting of

-   1,2-dipropionyl-sn-glycero-3-phosphocholine (03:0 PC),-   1,2-dibutyryl-sn-glycero-3-phosphocholine (04:0 PC),-   1,2-dipentanoyl-sn-glycero-3-phosphocholine (05:0 PC),-   1,2-dihexanoyl-sn-glycero-3-phosphocholine (06:0 PC),-   1,2-diheptanoyl-sn-glycero-3-phosphocholine (07:0 PC),-   1,2-dioctanoyl-sn-glycero-3-phosphocholine (08:0 PC),-   1,2-dinonanoyl-sn-glycero-3-phosphocholine (09:0 PC),-   1,2-didecanoyl-sn-glycero-3-phosphocholine (10:0 PC),-   1,2-diundecanoyl-sn-glycero-3-phosphocholine (11:0 PC, DUPC),-   1,2-dilauroyl-sn-glycero-3-phosphocholine (12:0 PC),-   1,2-ditridecanoyl-sn-glycero-3-phosphocholine (13:0 PC),-   1,2-dimyristoyl-sn-glycero-3-phosphocholine (14:0 PC, DMPC),-   1,2-dipentadecanoyl-sn-glycero-3-phosphocholine (15:0 PC),-   1,2-dipalmitoyl-sn-glycero-3-phosphocholine (16:0 PC, DPPC),-   1,2-diphytanoyl-sn-glycero-3-phosphocholine (4ME 16:0 PC),-   1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (17:0 PC),-   1,2-distearoyl-sn-glycero-3-phosphocholine (18:0 PC, DSPC),-   1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (19:0 PC),-   1,2-diarachidoyl-sn-glycero-3-phosphocholine (20:0 PC),-   1,2-dihenarachidoyl-sn-glycero-3-phosphocholine (21:0 PC),-   1,2-dibehenoyl-sn-glycero-3-phosphocholine (22:0 PC),-   1,2-ditricosanoyl-sn-glycero-3-phosphocholine (23:0 PC),-   1,2-dilignoceroyl-sn-glycero-3-phosphocholine (24:0 PC),-   1,2-dimyristoleoyl-sn-glycero-3-phosphocholine (14:1 (Δ9-Cis) PC),-   1,2-dimyristelaidoyl-sn-glycero-3-phosphocholine (14:1 (Δ9-Trans)    PC),-   1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (16:1 (Δ9-Cis) PC),-   1,2-dipalmitelaidoyl-sn-glycero-3-phosphocholine (16:1 (Δ9-Trans)    PC),-   1,2-dipetroselenoyl-sn-glycero-3-phosphocholine (18:1 (Δ6-Cis) PC),-   1,2-dioleoyl-sn-glycero-3-phosphocholine (18:1 (Δ9-Cis) PC, DOPC),-   1,2-dielaidoyl-sn-glycero-3-phosphocholine (18:1 (Δ9-Trans) PC),-   1,2-dilinoleoyl-sn-glycero-3-phosphocholine (18:2 (Cis) PC, DLPC),-   1,2-dilinolenoyl-sn-glycero-3-phosphocholine (18:3 (Cis) PC, DLnPC),-   1,2-dieicosenoyl-sn-glycero-3-phosphocholine (20:1 (Cis) PC),-   1,2-diarachidonoyl-sn-glycero-3-phosphocholine (20:4 (Cis) PC,    DAPC),-   1,2-dierucoyl-sn-glycero-3-phosphocholine (22:1 (Cis) PC),-   1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine (22:6 (Cis) PC,    DHAPC),-   1,2-dinervonoyl-sn-glycero-3-phosphocholine (24:1 (Cis) PC),-   1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine (06:0 PE),-   1,2-dioctanoyl-sn-glycero-3-phosphoethanolamine (08:0 PE),-   1,2-didecanoyl-sn-glycero-3-phosphoethanolamine (10:0 PE),-   1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (12:0 PE),-   1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (14:0 PE),-   1,2-dipentadecanoyl-sn-glycero-3-phosphoethanolamine (15:0 PE),-   1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (16:0 PE),-   1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (4ME 16:0 PE),-   1,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine (17:0 PE),-   1,2-di stearoyl-sn-glycero-3-phosphoethanolamine (18:0 PE, DSPE),-   1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (16:1 PE),-   1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (18:1 (Δ9-Cis) PE,    DOPE),-   1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (18:1 (Δ9-Trans)    PE),-   1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (18:2 PE, DLPE),-   1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine (18:3 PE, DLnPE),-   1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine (20:4 PE, DAPE),-   1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine (22:6 PE,    DHAPE),-   1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC),-   1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt    (DOPG), and

any combination thereof.

In some embodiments, the lipid composition of a pharmaceuticalcomposition disclosed herein comprises at least one symmetricphospholipid selected from the non-limiting group consisting of DLPC,DMPC, DOPC, DPPC, DSPC, DUPC, 18:0 Diether PC, DLnPC, DAPC, DHAPC, DOPE,4ME 16:0 PE, DSPE, DLPE, DLnPE, DAPE, DHAPE, DOPG, and any combinationthereof.

In some embodiments, the lipid composition of a pharmaceuticalcomposition disclosed herein comprises at least one asymmetricphospholipid. Asymmetric phospholipids can be selected from thenon-limiting group consisting of

-   1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (14:0-16:0 PC,    MPPC),-   1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (14:0-18:0 PC,    MSPC),-   1-palmitoyl-2-acetyl-sn-glycero-3-phosphocholine (16:0-02:0 PC),-   1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (16:0-14:0 PC,    PMPC),-   1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (16:0-18:0 PC,    PSPC),-   1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (16:0-18:1 PC,    POPC),-   1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (16:0-18:2 PC,    PLPC),-   1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (16:0-20:4    PC),-   1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (14:0-22:6    PC),-   1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (18:0-14:0 PC,    SMPC),-   1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (18:0-16:0 PC,    SPPC),-   1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0-18:1 PC,    SOPC),-   1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (18:0-18:2 PC),-   1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (18:0-20:4    PC),-   1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0-22:6    PC),-   1-oleoyl-2-myristoyl-sn-glycero-3-phosphocholine (18:1-14:0 PC,    OMPC),-   1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (18:1-16:0 PC,    OPPC),-   1-oleoyl-2-stearoyl-sn-glycero-3-phosphocholine (18:1-18:0 PC,    OSPC),-   1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1 PE,    POPE),-   1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:2    PE),-   1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine    (16:0-20:4 PE),-   1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine    (16:0-22:6 PE),-   1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (18:0-18:1 PE),-   1-stearoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (18:0-18:2    PE),-   1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine    (18:0-20:4 PE),-   1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine    (18:0-22:6 PE),-   1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine    (OChemsPC), and

any combination thereof.

Asymmetric lipids useful in the lipid composition can also belysolipids. Lysolipids can be selected from the non-limiting groupconsisting of

-   1-hexanoyl-2-hydroxy-sn-glycero-3-phosphocholine (06:0 Lyso PC),-   1-heptanoyl-2-hydroxy-sn-glycero-3-phosphocholine (07:0 Lyso PC),-   1-octanoyl-2-hydroxy-sn-glycero-3-phosphocholine (08:0 Lyso PC),-   1-nonanoyl-2-hydroxy-sn-glycero-3-phosphocholine (09:0 Lyso PC),-   1-decanoyl-2-hydroxy-sn-glycero-3-phosphocholine (10:0 Lyso PC),-   1-undecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (11:0 Lyso PC),-   1-lauroyl-2-hydroxy-sn-glycero-3-phosphocholine (12:0 Lyso PC),-   1-tridecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (13:0 Lyso PC),-   1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (14:0 Lyso PC),-   1-pentadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (15:0 Lyso    PC),-   1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (16:0 Lyso PC),-   1-heptadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (17:0 Lyso    PC),-   1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (18:0 Lyso PC),-   1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (18:1 Lyso PC),-   1-nonadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (19:0 Lyso PC),-   1-arachidoyl-2-hydroxy-sn-glycero-3-phosphocholine (20:0 Lyso PC),-   1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine (22:0 Lyso PC),-   1-lignoceroyl-2-hydroxy-sn-glycero-3-phosphocholine (24:0 Lyso PC),-   1-hexacosanoyl-2-hydroxy-sn-glycero-3-phosphocholine (26:0 Lyso PC),-   1-myristoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (14:0 Lyso    PE),-   1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (16:0 Lyso    PE),-   1-stearoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (18:0 Lyso    PE),-   1-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (18:1 Lyso PE),-   1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), and

any combination thereof.

In some embodiment, the lipid composition of a pharmaceuticalcomposition disclosed herein comprises at least one asymmetricphospholipid selected from the group consisting of MPPC, MSPC, PMPC,PSPC, SMPC, SPPC, and any combination thereof. In some embodiments, theasymmetric phospholipid is1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC).

In some embodiments, the lipid compositions disclosed herein can containone or more symmetric phospholipids, one or more asymmetricphospholipids, or a combination thereof. When multiple phospholipids arepresent, they can be present in equimolar ratios, or non-equimolarratios.

In one embodiment, the lipid composition of a pharmaceutical compositiondisclosed herein comprises a total amount of phospholipid (e.g., MSPC)which ranges from about 1 mol % to about 20 mol %, from about 5 mol % toabout 20 mol %, from about 10 mol % to about 20 mol %, from about 15 mol% to about 20 mol %, from about 1 mol % to about 15 mol %, from about 5mol % to about 15 mol %, from about 10 mol % to about 15 mol %, fromabout 5 mol % to about 10 mol % in the lipid composition. In oneembodiment, the amount of the phospholipid is from about 8 mol % toabout 15 mol % in the lipid composition. In one embodiment, the amountof the phospholipid (e.g., MSPC) is about 10 mol % in the lipidcomposition.

In some aspects, the amount of a specific phospholipid (e.g., MSPC) isat least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19 or 20 mol % in the lipid composition.

(ii) Quaternary Amine Compounds

The lipid composition of a pharmaceutical composition disclosed hereincan comprise one or more quaternary amine compounds (e.g., DOTAP). Theterm “quaternary amine compound” is used to include those compoundshaving one or more quaternary amine groups (e.g., trialkylamino groups)and permanently carrying a positive charge and existing in a form of asalt. For example, the one or more quaternary amine groups can bepresent in a lipid or a polymer (e.g., PEG). In some embodiments, thequaternary amine compound comprises (1) a quaternary amine group and (2)at least one hydrophobic tail group comprising (i) a hydrocarbon chain,linear or branched, and saturated or unsaturated, and (ii) optionally anether, ester, carbonyl, or ketal linkage between the quaternary aminegroup and the hydrocarbon chain. In some embodiments, the quaternaryamine group can be a trimethylammonium group. In some embodiments, thequaternary amine compound comprises two identical hydrocarbon chains. Insome embodiments, the quaternary amine compound comprises two differenthydrocarbon chains.

In some embodiments, the lipid composition of a pharmaceuticalcomposition disclosed herein comprises at least one quaternary aminecompound. Quaternary amine compound can be selected from thenon-limiting group consisting of

-   1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),-   N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride    (DOTMA),-   1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium    chloride (DOTIM),-   2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium    trifluoroacetate (DOSPA),-   N,N-distearyl-N,N-dimethylammonium bromide (DDAB),-   N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium    bromide (DMRIE),-   N-(1,2-dioleoyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium    bromide (DORIE),-   N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),-   1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLePC),-   1,2-distearoyl-3-trimethylammonium-propane (DSTAP),-   1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP),-   1,2-dilinoleoyl-3-trimethylammonium-propane (DLTAP),-   1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP)-   1,2-distearoyl-sn-glycero-3-ethylphosphocholine (DSePC)-   1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (DPePC),-   1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMePC),-   1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOePC),-   1,2-di-(9Z-tetradecenoyl)-sn-glycero-3-ethylphosphocholine (14:1    EPC),-   1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:0-18:1    EPC),

and any combination thereof.

In one embodiment, the quaternary amine compound is1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).

Quaternary amine compounds are known in the art, such as those describedin US 2013/0245107 A1, US 2014/0363493 A1, U.S. Pat. No. 8,158,601, WO2015/123264 A1, and WO 2015/148247 A1, which are incorporated herein byreference in their entirety.

In one embodiment, the amount of the quaternary amine compound (e.g.,DOTAP) in the lipid composition disclosed herein ranges from about 0.01mol % to about 20 mol %.

In one embodiment, the amount of the quaternary amine compound (e.g.,DOTAP) in the lipid composition disclosed herein ranges from about 0.5mol % to about 20 mol %, from about 0.5 mol % to about 15 mol %, fromabout 0.5 mol % to about 10 mol %, from about 1 mol % to about 20 mol %,from about 1 mol % to about 15 mol %, from about 1 mol % to about 10 mol%, from about 2 mol % to about 20 mol %, from about 2 mol % to about 15mol %, from about 2 mol % to about 10 mol %, from about 3 mol % to about20 mol %, from about 3 mol % to about 15 mol %, from about 3 mol % toabout 10 mol %, from about 4 mol % to about 20 mol %, from about 4 mol %to about 15 mol %, from about 4 mol % to about 10 mol %, from about 5mol % to about 20 mol %, from about 5 mol % to about 15 mol %, fromabout 5 mol % to about 10 mol %, from about 6 mol % to about 20 mol %,from about 6 mol % to about 15 mol %, from about 6 mol % to about 10 mol%, from about 7 mol % to about 20 mol %, from about 7 mol % to about 15mol %, from about 7 mol % to about 10 mol %, from about 8 mol % to about20 mol %, from about 8 mol % to about 15 mol %, from about 8 mol % toabout 10 mol %, from about 9 mol % to about 20 mol %, from about 9 mol %to about 15 mol %, from about 9 mol % to about 10 mol %.

In one embodiment, the amount of the quaternary amine compound (e.g.,DOTAP) in the lipid composition disclosed herein ranges from about 5 mol% to about 10 mol %.

In one embodiment, the amount of the quaternary amine compound (e.g.,DOTAP) in the lipid composition disclosed herein is about 5 mol %. Inone embodiment, the amount of the quaternary amine compound (e.g.,DOTAP) in the lipid composition disclosed herein is about 10 mol %.

In some embodiments, the amount of the quaternary amine compound (e.g.,DOTAP) is at least about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08,0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3,3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5,12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5,19, 19.5 or 20 mol % in the lipid composition disclosed herein.

In one embodiment, the mole ratio of the compound of formula (I) (e.g.,Compounds 18, 25, 26 or 48) to the quaternary amine compound (e.g.,DOTA) is about 100:1 to about 2.5:1. In one embodiment, the mole ratioof the compound of formula (I) (e.g., Compounds 18, 25, 26 or 48) to thequaternary amine compound (e.g., DOTAP) is about 90:1, about 80:1, about70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 20:1, about15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1,or about 2.5:1. In one embodiment, the mole ratio of the compound offormula (I) (e.g., Compounds 18, 25, 26 or 48) to the quaternary aminecompound (e.g., DOTAP) in the lipid composition disclosed herein isabout 10:1.

In some aspects, the lipid composition the pharmaceutical compositionsdisclosed herein does not comprise a quaternary amine compound. In someaspects, the lipid composition of the pharmaceutical compositionsdisclosed does not comprise DOTAP.

(iii) Structural Lipids

The lipid composition of a pharmaceutical composition disclosed hereincan comprise one or more structural lipids. As used herein, the term“structural lipid” refers to sterols and also to lipids containingsterol moieties. In some embodiments, the structural lipid is selectedfrom the group consisting of cholesterol, fecosterol, sitosterol,ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine,tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof. In someembodiments, the structural lipid is cholesterol.

In one embodiment, the amount of the structural lipid (e.g., an sterolsuch as cholesterol) in the lipid composition of a pharmaceuticalcomposition disclosed herein ranges from about 20 mol % to about 60 mol%, from about 25 mol % to about 55 mol %, from about 30 mol % to about50 mol %, or from about 35 mol % to about 45 mol %.

In one embodiment, the amount of the structural lipid (e.g., an sterolsuch as cholesterol) in the lipid composition disclosed herein rangesfrom about 25 mol % to about 30 mol %, from about 30 mol % to about 35mol %, or from about 35 mol % to about 40 mol %.

In one embodiment, the amount of the structural lipid (e.g., a sterolsuch as cholesterol) in the lipid composition disclosed herein is about23.5 mol %, about 28.5 mol %, about 33.5 mol %, or about 38.5 mol %.

In some embodiments, the amount of the structural lipid (e.g., an sterolsuch as cholesterol) in the lipid composition disclosed herein is atleast about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, or 60 mol %.

In some aspects, the lipid composition component of the pharmaceuticalcompositions for intratumoral delivery disclosed does not comprisecholesterol.

(iv) Polyethylene Glycol (PEG)-Lipids

The lipid composition of a pharmaceutical composition disclosed hereincan comprise one or more a polyethylene glycol (PEG) lipid.

As used herein, the term “PEG-lipid” refers to polyethylene glycol(PEG)-modified lipids. Non-limiting examples of PEG-lipids includePEG-modified phosphatidylethanolamine and phosphatidic acid,PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modifieddialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines. Such lipidsare also referred to as PEGylated lipids. For example, a PEG lipid canbe PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPElipid.

In some embodiments, the PEG-lipid includes, but not limited to1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl,PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG),PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), orPEG-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA).

In one embodiment, the PEG-lipid is selected from the group consistingof a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidicacid, a PEG-modified ceramide, a PEG-modified dialkylamine, aPEG-modified diacylglycerol, a PEG-modified dialkylglycerol, andmixtures thereof.

In some embodiments, the lipid moiety of the PEG-lipids includes thosehaving lengths of from about C₁₄ to about C₂₂, preferably from about C₁₄to about C₁₆. In some embodiments, a PEG moiety, for example anmPEG-NH₂, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000daltons. In one embodiment, the PEG-lipid is PEG_(2k)-DMG.

In one embodiment, the lipid nanoparticles described herein can comprisea PEG lipid which is a non-diffusible PEG. Non-limiting examples ofnon-diffusible PEGs include PEG-DSG and PEG-DSPE.

PEG-lipids are known in the art, such as those described in U.S. Pat.No. 8,158,601 and International Publ. No. WO 2015/130584 A2, which areincorporated herein by reference in their entirety.

In one embodiment, the amount of PEG-lipid in the lipid composition of apharmaceutical composition disclosed herein ranges from about 0.1 mol %to about 5 mol %, from about 0.5 mol % to about 5 mol %, from about 1mol % to about 5 mol %, from about 1.5 mol % to about 5 mol %, fromabout 2 mol % to about 5 mol % mol %, from about 0.1 mol % to about 4mol %, from about 0.5 mol % to about 4 mol %, from about 1 mol % toabout 4 mol %, from about 1.5 mol % to about 4 mol %, from about 2 mol %to about 4 mol %, from about 0.1 mol % to about 3 mol %, from about 0.5mol % to about 3 mol %, from about 1 mol % to about 3 mol %, from about1.5 mol % to about 3 mol %, from about 2 mol % to about 3 mol %, fromabout 0.1 mol % to about 2 mol %, from about 0.5 mol % to about 2 mol %,from about 1 mol % to about 2 mol %, from about 1.5 mol % to about 2 mol%, from about 0.1 mol % to about 1.5 mol %, from about 0.5 mol % toabout 1.5 mol %, or from about 1 mol % to about 1.5 mol %.

In one embodiment, the amount of PEG-lipid in the lipid compositiondisclosed herein is about 1.5 mol %.

In one embodiment, the amount of PEG-lipid in the lipid compositiondisclosed herein is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5 mol %.

In some aspects, the lipid composition of the pharmaceuticalcompositions disclosed herein does not comprise a PEG-lipid.

In some embodiments, the lipid composition disclosed herein comprises acompound of formula (I) and an asymmetric phospholipid. In someembodiments, the lipid composition comprises compound 18 and MSPC.

In some embodiments, the lipid composition disclosed herein comprises acompound of formula (I) and a quaternary amine compound. In someembodiments, the lipid composition comprises compound 18 and DOTAP.

In some embodiments, the lipid composition disclosed herein comprises acompound of formula (I), an asymmetric phospholipid, and a quaternaryamine compound. In some embodiments, the lipid composition comprisescompound 18, MSPC and DOTAP.

In one embodiment, the lipid composition comprises about 50 mol % of acompound of formula (I) (e.g., Compounds 18, 25, 26 or 48), about 10 mol% of DSPC or MSPC, about 33.5 mol % of cholesterol, about 1.5 mol % ofPEG-DMG, and about 5 mol % of DOTAP. In one embodiment, the lipidcomposition comprises about 50 mol % of a compound of formula (I) (e.g.Compounds 18, 25, 26 or 48), about 10 mol % of DSPC or MSPC, about 28.5mol % of cholesterol, about 1.5 mol % of PEG-DMG, and about 10 mol % ofDOTAP.

The components of the lipid nanoparticle can be tailored for optimaldelivery of the polynucleotides based on the desired outcome. As anon-limiting example, the lipid nanoparticle can comprise 40-60 mol % acompound of formula (I), 8-16 mol % phospholipid, 30-45 mol %cholesterol, 1-5 mol % PEG lipid, and optionally 1-15 mol % quaternaryamine compound.

In some embodiments, the lipid nanoparticle can comprise 45-65 mol % ofa compound of formula (I), 5-10 mol % phospholipid, 25-40 mol %cholesterol, 0.5-5 mol % PEG lipid, and optionally 1-15 mol % quaternaryamine compound.

Non-limiting examples of nucleic acid lipid particles are disclosed inU.S. Patent Publication No. 20140121263, herein incorporated byreference in its entirety.

(v) Other Ionizable Amino Lipids

The lipid composition of the pharmaceutical composition disclosed hereincan comprise one or more ionizable amino lipids in addition to a lipidaccording to formula (I).

Ionizable lipids can be selected from the non-limiting group consistingof 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10),N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine(KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25),1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate(DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),(13Z,165Z)—N,N-dimethyl-3-nonydocosa-13-16-dien-1-amine (L608),2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA),(2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,2-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2R)), and(2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2S)). In addition to these, an ionizable amino lipid canalso be a lipid including a cyclic amine group.

Ionizable lipids can also be the compounds disclosed in InternationalPublication No. WO 2015/199952 A1, hereby incorporated by reference inits entirety. For example, the ionizable amino lipids include, but notlimited to:

and any combination thereof.

(vi) Other Lipid Composition Components

The lipid composition of a pharmaceutical composition disclosed hereincan include one or more components in addition to those described above.For example, the lipid composition can include one or more permeabilityenhancer molecules, carbohydrates, polymers, surface altering agents(e.g., surfactants), or other components. For example, a permeabilityenhancer molecule can be a molecule described by U.S. Patent ApplicationPublication No. 2005/0222064. Carbohydrates can include simple sugars(e.g., glucose) and polysaccharides (e.g., glycogen and derivatives andanalogs thereof). The lipid composition can include a buffer such as,but not limited to, citrate or phosphate at a pH of 7, salt and/orsugar. Salt and/or sugar can be included in the formulations describedherein for isotonicity.

A polymer can be included in and/or used to encapsulate or partiallyencapsulate a pharmaceutical composition disclosed herein (e.g., apharmaceutical composition in lipid nanoparticle form). A polymer can bebiodegradable and/or biocompatible. A polymer can be selected from, butis not limited to, polyamines, polyethers, polyamides, polyesters,polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides,polysulfones, polyurethanes, polyacetylenes, polyethylenes,polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates,polyacrylonitriles, and polyarylates.

The ratio between the lipid composition and the polynucleotide range canbe from about 10:1 to about 60:1 (wt/wt).

In some embodiments, the ratio between the lipid composition and thepolynucleotide can be about 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1,17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1,29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1,41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1,53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1 or 60:1 (wt/wt). In someembodiments, the wt/wt ratio of the lipid composition to thepolynucleotide encoding a therapeutic agent is about 20:1 or about 15:1.

In some embodiments, the pharmaceutical composition disclosed herein cancontain more than one polypeptides. For example, a pharmaceuticalcomposition disclosed herein can contain two or more polynucleotides(e.g., RNA, e.g., mRNA).

In one embodiment, the lipid nanoparticles described herein can comprisepolynucleotides (e.g., mRNA) in a lipid:polynucleotide weight ratio of5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1 or70:1, or a range or any of these ratios such as, but not limited to, 5:1to about 10:1, from about 5:1 to about 15:1, from about 5:1 to about20:1, from about 5:1 to about 25:1, from about 5:1 to about 30:1, fromabout 5:1 to about 35:1, from about 5:1 to about 40:1, from about 5:1 toabout 45:1, from about 5:1 to about 50:1, from about 5:1 to about 55:1,from about 5:1 to about 60:1, from about 5:1 to about 70:1, from about10:1 to about 15:1, from about 10:1 to about 20:1, from about 10:1 toabout 25:1, from about 10:1 to about 30:1, from about 10:1 to about35:1, from about 10:1 to about 40:1, from about 10:1 to about 45:1, fromabout 10:1 to about 50:1, from about 10:1 to about 55:1, from about 10:1to about 60:1, from about 10:1 to about 70:1, from about 15:1 to about20:1, from about 15:1 to about 25:1, from about 15:1 to about 30:1, fromabout 15:1 to about 35:1, from about 15:1 to about 40:1, from about 15:1to about 45:1, from about 15:1 to about 50:1, from about 15:1 to about55:1, from about 15:1 to about 60:1 or from about 15:1 to about 70:1.

In one embodiment, the lipid nanoparticles described herein can comprisethe polynucleotide in a concentration from approximately 0.1 mg/ml to 2mg/ml such as, but not limited to, 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml,1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 1.7mg/ml, 1.8 mg/ml, 1.9 mg/ml, 2.0 mg/ml or greater than 2.0 mg/ml.

In one embodiment, formulations comprising the polynucleotides and lipidnanoparticles described herein can comprise 0.15 mg/ml to 2 mg/ml of thepolynucleotide described herein (e.g., mRNA). In some embodiments, theformulation can further comprise 10 mM of citrate buffer and theformulation can additionally comprise up to 10% w/w of sucrose (e.g., atleast 1% w/w, at least 2% w/w/, at least 3% w/w, at least 4% w/w, atleast 5% w/w, at least 6% w/w, at least 7% w/w, at least 8% w/w, atleast 9% w/w or 10% w/w).

(vii) Nanoparticle Compositions

In some embodiments, the pharmaceutical compositions disclosed hereinare formulated as lipid nanoparticles (LNP). Accordingly, the presentdisclosure also provides nanoparticle compositions comprising (i) alipid composition comprising a compound of formula (I) as describedherein, and (ii) a polynucleotide encoding an MCM polypeptide. In suchnanoparticle composition, the lipid composition disclosed herein canencapsulate the polynucleotide encoding an MCM polypeptide.

Nanoparticle compositions are typically sized on the order ofmicrometers or smaller and can include a lipid bilayer. Nanoparticlecompositions encompass lipid nanoparticles (LNPs), liposomes (e.g.,lipid vesicles), and lipoplexes. For example, a nanoparticle compositioncan be a liposome having a lipid bilayer with a diameter of 500 nm orless.

Nanoparticle compositions include, for example, lipid nanoparticles(LNPs), liposomes, and lipoplexes. In some embodiments, nanoparticlecompositions are vesicles including one or more lipid bilayers. Incertain embodiments, a nanoparticle composition includes two or moreconcentric bilayers separated by aqueous compartments. Lipid bilayerscan be functionalized and/or crosslinked to one another. Lipid bilayerscan include one or more ligands, proteins, or channels.

Nanoparticle compositions of the present disclosure comprise at leastone compound according to formula (I). For example, the nanoparticlecomposition can include one or more of Compounds 1-147. Nanoparticlecompositions can also include a variety of other components. Forexample, the nanoparticle composition can include one or more otherlipids in addition to a lipid according to formula (I) or (II), forexample (i) at least one phospholipid, (ii) at least one quaternaryamine compound, (iii) at least one structural lipid, (iv) at least onePEG-lipid, or (v) any combination thereof.

In some embodiments, the nanoparticle composition comprises a compoundof formula (I), (e.g., Compounds 18, 25, 26 or 48). In some embodiments,the nanoparticle composition comprises a compound of formula (I) (e.g.,Compounds 18, 25, 26 or 48) and a phospholipid (e.g., DSPC or MSPC). Insome embodiments, the nanoparticle composition comprises a compound offormula (I) (e.g., Compounds 18, 25, 26 or 48), a phospholipid (e.g.,DSPC or MSPC), and a quaternary amine compound (e.g., DOTAP). In someembodiments, the nanoparticle composition comprises a compound offormula (I) (e.g., Compounds 18, 25, 26 or 48), and a quaternary aminecompound (e.g., DOTAP).

In some embodiments, the nanoparticle composition comprises a lipidcomposition consisting or consisting essentially of compound of formula(I) (e.g., Compounds 18, 25, 26 or 48). In some embodiments, thenanoparticle composition comprises a lipid composition consisting orconsisting essentially of a compound of formula (I) (e.g., Compounds 18,25, 26 or 48) and a phospholipid (e.g., DSPC or MSPC). In someembodiments, the nanoparticle composition comprises a lipid compositionconsisting or consisting essentially of a compound of formula (I) (e.g.,Compounds 18, 25, 26 or 48), a phospholipid (e.g., DSPC or MSPC), and aquaternary amine compound (e.g., DOTAP). In some embodiments, thenanoparticle composition comprises a lipid composition consisting orconsisting essentially of a compound of formula (I) (e.g., Compounds 18,25, 26 or 48), and a quaternary amine compound (e.g., DOTAP).

Nanoparticle compositions can be characterized by a variety of methods.For example, microscopy (e.g., transmission electron microscopy orscanning electron microscopy) can be used to examine the morphology andsize distribution of a nanoparticle composition. Dynamic lightscattering or potentiometry (e.g., potentiometric titrations) can beused to measure zeta potentials. Dynamic light scattering can also beutilized to determine particle sizes. Instruments such as the ZetasizerNano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can alsobe used to measure multiple characteristics of a nanoparticlecomposition, such as particle size, polydispersity index, and zetapotential.

The size of the nanoparticles can help counter biological reactions suchas, but not limited to, inflammation, or can increase the biologicaleffect of the polynucleotide.

As used herein, “size” or “mean size” in the context of nanoparticlecompositions refers to the mean diameter of a nanoparticle composition.

In one embodiment, the polynucleotide encoding an MCM polypeptide areformulated in lipid nanoparticles having a diameter from about 10 toabout 100 nm such as, but not limited to, about 10 to about 20 nm, about10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm,about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 toabout 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm,about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 toabout 60 nm, about 50 to about 70 nm, about 50 to about 80 nm, about 50to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm,about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100nm, about 80 to about 90 nm, about 80 to about 100 nm and/or about 90 toabout 100 nm.

In one embodiment, the nanoparticles have a diameter from about 10 to500 nm. In one embodiment, the nanoparticle has a diameter greater than100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm,greater than 300 nm, greater than 350 nm, greater than 400 nm, greaterthan 450 nm, greater than 500 nm, greater than 550 nm, greater than 600nm, greater than 650 nm, greater than 700 nm, greater than 750 nm,greater than 800 nm, greater than 850 nm, greater than 900 nm, greaterthan 950 nm or greater than 1000 nm.

In some embodiments, the largest dimension of a nanoparticle compositionis 1 μm or shorter (e.g., 1 μm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm,400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, orshorter).

A nanoparticle composition can be relatively homogenous. Apolydispersity index can be used to indicate the homogeneity of ananoparticle composition, e.g., the particle size distribution of thenanoparticle composition. A small (e.g., less than 0.3) polydispersityindex generally indicates a narrow particle size distribution. Ananoparticle composition can have a polydispersity index from about 0 toabout 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08,0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20,0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersityindex of a nanoparticle composition disclosed herein can be from about0.10 to about 0.20.

The zeta potential of a nanoparticle composition can be used to indicatethe electrokinetic potential of the composition. For example, the zetapotential can describe the surface charge of a nanoparticle composition.Nanoparticle compositions with relatively low charges, positive ornegative, are generally desirable, as more highly charged species caninteract undesirably with cells, tissues, and other elements in thebody. In some embodiments, the zeta potential of a nanoparticlecomposition disclosed herein can be from about −10 mV to about +20 mV,from about −10 mV to about +15 mV, from about 10 mV to about +10 mV,from about −10 mV to about +5 mV, from about −10 mV to about 0 mV, fromabout −10 mV to about −5 mV, from about −5 mV to about +20 mV, fromabout −5 mV to about +15 mV, from about −5 mV to about +10 mV, fromabout −5 mV to about +5 mV, from about −5 mV to about 0 mV, from about 0mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV toabout +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about+20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about+10 mV.

In some embodiments, the zeta potential of the lipid nanoparticles canbe from about 0 mV to about 100 mV, from about 0 mV to about 90 mV, fromabout 0 mV to about 80 mV, from about 0 mV to about 70 mV, from about 0mV to about 60 mV, from about 0 mV to about 50 mV, from about 0 mV toabout 40 mV, from about 0 mV to about 30 mV, from about 0 mV to about 20mV, from about 0 mV to about 10 mV, from about 10 mV to about 100 mV,from about 10 mV to about 90 mV, from about 10 mV to about 80 mV, fromabout 10 mV to about 70 mV, from about 10 mV to about 60 mV, from about10 mV to about 50 mV, from about 10 mV to about 40 mV, from about 10 mVto about 30 mV, from about 10 mV to about 20 mV, from about 20 mV toabout 100 mV, from about 20 mV to about 90 mV, from about 20 mV to about80 mV, from about 20 mV to about 70 mV, from about 20 mV to about 60 mV,from about 20 mV to about 50 mV, from about 20 mV to about 40 mV, fromabout 20 mV to about 30 mV, from about 30 mV to about 100 mV, from about30 mV to about 90 mV, from about 30 mV to about 80 mV, from about 30 mVto about 70 mV, from about 30 mV to about 60 mV, from about 30 mV toabout 50 mV, from about 30 mV to about 40 mV, from about 40 mV to about100 mV, from about 40 mV to about 90 mV, from about 40 mV to about 80mV, from about 40 mV to about 70 mV, from about 40 mV to about 60 mV,and from about 40 mV to about 50 mV. In some embodiments, the zetapotential of the lipid nanoparticles can be from about 10 mV to about 50mV, from about 15 mV to about 45 mV, from about 20 mV to about 40 mV,and from about 25 mV to about 35 mV. In some embodiments, the zetapotential of the lipid nanoparticles can be about 10 mV, about 20 mV,about 30 mV, about 40 mV, about 50 mV, about 60 mV, about 70 mV, about80 mV, about 90 mV, and about 100 mV.

The term “encapsulation efficiency” of a polynucleotide describes theamount of the polynucleotide that is encapsulated by or otherwiseassociated with a nanoparticle composition after preparation, relativeto the initial amount provided. As used herein, “encapsulation” canrefer to complete, substantial, or partial enclosure, confinement,surrounding, or encasement.

Encapsulation efficiency is desirably high (e.g., close to 100%). Theencapsulation efficiency can be measured, for example, by comparing theamount of the polynucleotide in a solution containing the nanoparticlecomposition before and after breaking up the nanoparticle compositionwith one or more organic solvents or detergents.

Fluorescence can be used to measure the amount of free polynucleotide ina solution. For the nanoparticle compositions described herein, theencapsulation efficiency of a polynucleotide can be at least 50%, forexample 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulationefficiency can be at least 80%. In certain embodiments, theencapsulation efficiency can be at least 90%.

The amount of a polynucleotide present in a pharmaceutical compositiondisclosed herein can depend on multiple factors such as the size of thepolynucleotide, desired target and/or application, or other propertiesof the nanoparticle composition as well as on the properties of thepolynucleotide.

For example, the amount of an mRNA useful in a nanoparticle compositioncan depend on the size (expressed as length, or molecular mass),sequence, and other characteristics of the mRNA. The relative amounts ofa polynucleotide in a nanoparticle composition can also vary.

The relative amounts of the lipid composition and the polynucleotidepresent in a lipid nanoparticle composition of the present disclosurecan be optimized according to considerations of efficacy andtolerability. For compositions including an mRNA as a polynucleotide,the N:P ratio can serve as a useful metric.

As the N:P ratio of a nanoparticle composition controls both expressionand tolerability, nanoparticle compositions with low N:P ratios andstrong expression are desirable. N:P ratios vary according to the ratioof lipids to RNA in a nanoparticle composition.

In general, a lower N:P ratio is preferred. The one or more RNA, lipids,and amounts thereof can be selected to provide an N:P ratio from about2:1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1,12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. Incertain embodiments, the N:P ratio can be from about 2:1 to about 8:1.In other embodiments, the N:P ratio is from about 5:1 to about 8:1. Incertain embodiments, the N:P ratio is between 5:1 and 6:1. In onespecific aspect, the N:P ratio is about is about 5.67:1.

In addition to providing nanoparticle compositions, the presentdisclosure also provides methods of producing lipid nanoparticlescomprising encapsulating a polynucleotide. Such method comprises usingany of the pharmaceutical compositions disclosed herein and producinglipid nanoparticles in accordance with methods of production of lipidnanoparticles known in the art. See, e.g., Wang et al. (2015) “Deliveryof oligonucleotides with lipid nanoparticles” Adv. Drug Deliv. Rev.87:68-80; Silva et al. (2015) “Delivery Systems for Biopharmaceuticals.Part I: Nanoparticles and Microparticles” Curr. Pharm. Technol. 16:940-954; Naseri et al. (2015) “Solid Lipid Nanoparticles andNanostructured Lipid Carriers: Structure, Preparation and Application”Adv. Pharm. Bull. 5:305-13; Silva et al. (2015) “Lipid nanoparticles forthe delivery of biopharmaceuticals” Curr. Pharm. Biotechnol. 16:291-302,and references cited therein.

Excipients

The disclosure also includes pharmaceutical compositions that comprise aformulation of a polynucleotide described herein, i.e., a polynucleotidecomprising an ORF encoding an MCM polypeptide, with excipients. Thepolynucleotides of the disclosure can be formulated using one or moreexcipients to: (1) increase stability; (2) increase cell transfection;(3) permit the sustained or delayed release (e.g., from a depotformulation of the polynucleotide); (4) alter the biodistribution (e.g.,target the polynucleotide to specific tissues or cell types); (5)increase the translation of encoded protein in vivo; and/or (6) alterthe release profile of encoded protein in vivo. In addition totraditional excipients such as any and all solvents, dispersion media,diluents, or other liquid vehicles, dispersion or suspension aids,surface active agents, isotonic agents, thickening or emulsifyingagents, preservatives, excipients of the present disclosure can include,without limitation, lipidoids, liposomes, lipid nanoparticles, polymers,lipoplexes, core-shell nanoparticles, peptides, proteins, cellstransfected with polynucleotides (e.g., for transplantation into asubject), hyaluronidase, nanoparticle mimics and combinations thereof.Accordingly, the formulations of the disclosure can include one or moreexcipients, each in an amount that together increases the stability ofthe polynucleotide, increases cell transfection by the polynucleotide,increases the expression of polynucleotides encoded protein, and/oralters the release profile of polynucleotide encoded proteins. Further,the polynucleotides of the present disclosure can be formulated usingself-assembled nucleic acid nanoparticles.

A pharmaceutically acceptable excipient, as used herein, includes, butare not limited to, any and all solvents, dispersion media, diluents, orother liquid vehicles, dispersion or suspension aids, surface activeagents, isotonic agents, thickening or emulsifying agents,preservatives, solid binders, lubricants, flavoring agents, stabilizers,antioxidants, osmolality adjusting agents, pH adjusting agents and thelike, as suited to the particular dosage form desired. Variousexcipients for formulating pharmaceutical compositions and techniquesfor preparing the composition are known in the art (see Remington: TheScience and Practice of Pharmacy, 21^(st) Edition, A. R. Gennaro(Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporatedherein by reference in its entirety). The use of a conventionalexcipient medium can be contemplated within the scope of the presentdisclosure, except insofar as any conventional excipient medium isincompatible with a substance or its derivatives, such as by producingany undesirable biological effect or otherwise interacting in adeleterious manner with any other component(s) of the pharmaceuticalcomposition, its use is contemplated to be within the scope of thisdisclosure.

In some embodiments, a pharmaceutically acceptable excipient can be atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% pure. In some embodiments, an excipient is approved for use forhumans and for veterinary use. In some embodiments, an excipient can beapproved by United States Food and Drug Administration. In someembodiments, an excipient can be of pharmaceutical grade. In someembodiments, an excipient can meet the standards of the United StatesPharmacopoeia (USP), the European Pharmacopoeia (EP), the BritishPharmacopoeia, and/or the International Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, dispersing and/or granulating agents, surface active agentsand/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils. Suchexcipients can optionally be included in pharmaceutical compositions.The composition can also include excipients such as cocoa butter andsuppository waxes, coloring agents, coating agents, sweetening,flavoring, and/or perfuming agents.

Exemplary diluents include, but are not limited to, calcium carbonate,sodium carbonate, calcium phosphate, dicalcium phosphate, calciumsulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose,cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc.,and/or combinations thereof.

Exemplary granulating and/or dispersing agents include, but are notlimited to, potato starch, corn starch, tapioca starch, sodium starchglycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite,cellulose and wood products, natural sponge, cation-exchange resins,calcium carbonate, silicates, sodium carbonate, cross-linkedpoly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch(sodium starch glycolate), carboxymethyl cellulose, cross-linked sodiumcarboxymethyl cellulose (croscarmellose), methylcellulose,pregelatinized starch (starch 1500), microcrystalline starch, waterinsoluble starch, calcium carboxymethyl cellulose, magnesium aluminumsilicate (VEEGUM®), sodium lauryl sulfate, quaternary ammoniumcompounds, etc., and/or combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are notlimited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodiumalginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin,egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidalclays (e.g., bentonite [aluminum silicate] and VEEGUM® [magnesiumaluminum silicate]), long chain amino acid derivatives, high molecularweight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol,triacetin monostearate, ethylene glycol distearate, glycerylmonostearate, and propylene glycol monostearate, polyvinyl alcohol),carbomers (e.g., carboxy polymethylene, polyacrylic acid, acrylic acidpolymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives(e.g., carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylenesorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEEN®60],polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate[SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate[SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]),polyoxyethylene esters (e.g., polyoxyethylene monostearate [MYRJ®45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g., CREMOPHOR®),polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether [BRIJ®30]),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, PLUORINC®F 68, POLOXAMER®188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include, but are not limited to, starch (e.g.,cornstarch and starch paste); gelatin; sugars (e.g., sucrose, glucose,dextrose, dextrin, molasses, lactose, lactitol, mannitol); amino acids(e.g., glycine); natural and synthetic gums (e.g., acacia, sodiumalginate, extract of Irish moss, panwar gum, ghatti gum, mucilage ofisapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate,poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM®), andlarch arabogalactan); alginates; polyethylene oxide; polyethyleneglycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes;water; alcohol; etc.; and combinations thereof.

Exemplary preservatives can include, but are not limited to,antioxidants, chelating agents, antimicrobial preservatives, antifungalpreservatives, alcohol preservatives, acidic preservatives, and/or otherpreservatives. Oxidation is a potential degradation pathway for mRNA,especially for liquid mRNA formulations. In order to prevent oxidation,antioxidants can be added to the formulation. Exemplary antioxidantsinclude, but are not limited to, alpha tocopherol, ascorbic acid,acorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, EDTA,m-cresol, methionine, butylated hydroxytoluene, monothioglycerol,potassium metabisulfite, propionic acid, propyl gallate, sodiumascorbate, sodium bisulfite, sodium metabisulfite, thioglycerol and/orsodium sulfite. Exemplary chelating agents includeethylenediaminetetraacetic acid (EDTA), citric acid monohydrate,disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malicacid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodiumedetate. Exemplary antimicrobial preservatives include, but are notlimited to, benzalkonium chloride, benzethonium chloride, benzylalcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine,chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol,glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethylalcohol, phenylmercuric nitrate, propylene glycol, and/or thimerosal.Exemplary antifungal preservatives include, but are not limited to,butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoicacid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodiumbenzoate, sodium propionate, and/or sorbic acid. Exemplary alcoholpreservatives include, but are not limited to, ethanol, polyethyleneglycol, phenol, phenolic compounds, bisphenol, chlorobutanol,hydroxybenzoate, and/or phenylethyl alcohol. Exemplary acidicpreservatives include, but are not limited to, vitamin A, vitamin C,vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid,ascorbic acid, sorbic acid, and/or phytic acid. Other preservativesinclude, but are not limited to, tocopherol, tocopherol acetate,deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylatedhydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS),sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, GLYDANTPLUS®, PHENONIP®, methylparaben, GERMALL® 15, GERMABEN®II, NEOLONE™,KATHON™, and/or EUXYL®.

In some embodiments, the pH of polynucleotide solutions are maintainedbetween pH 5 and pH 8 to improve stability. Exemplary buffers to controlpH can include, but are not limited to sodium phosphate, sodium citrate,sodium succinate, histidine (or histidine-HCl), sodium carbonate, and/orsodium malate. In another embodiment, the exemplary buffers listed abovecan be used with additional monovalent counterions (including, but notlimited to potassium). Divalent cations can also be used as buffercounterions; however, these are subject to complex formation and/or mRNAdegradation.

Exemplary buffering agents can also include, but are not limited to,citrate buffer solutions, acetate buffer solutions, phosphate buffersolutions, ammonium chloride, calcium carbonate, calcium chloride,calcium citrate, calcium glubionate, calcium gluceptate, calciumgluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate,propanoic acid, calcium levulinate, pentanoic acid, dibasic calciumphosphate, phosphoric acid, tribasic calcium phosphate, calciumhydroxide phosphate, potassium acetate, potassium chloride, potassiumgluconate, potassium mixtures, dibasic potassium phosphate, monobasicpotassium phosphate, potassium phosphate mixtures, sodium acetate,sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate,dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphatemixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginicacid, pyrogen-free water, isotonic saline, Ringer's solution, ethylalcohol, etc., and/or combinations thereof.

Exemplary lubricating agents include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, silica, talc, malt, glycerylbehanate, hydrogenated vegetable oils, polyethylene glycol, sodiumbenzoate, sodium acetate, sodium chloride, leucine, magnesium laurylsulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include, but are not limited to, almond, apricot kernel,avocado, babassu, bergamot, black current seed, borage, cade, camomile,canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, codliver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose,fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop,isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon,litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink,nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel,peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary,safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, sheabutter, silicone, soybean, sunflower, tea tree, thistle, tsubaki,vetiver, walnut, and wheat germ oils. Exemplary oils include, but arenot limited to, butyl stearate, caprylic triglyceride, caprictriglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,silicone oil, and/or combinations thereof.

Excipients such as cocoa butter and suppository waxes, coloring agents,coating agents, sweetening, flavoring, and/or perfuming agents can bepresent in the composition, according to the judgment of the formulator.

Exemplary additives include physiologically biocompatible buffers (e.g.,trimethylamine hydrochloride), addition of chelants (such as, forexample, DTPA or DTPA-bisamide) or calcium chelate complexes (as forexample calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions ofcalcium or sodium salts (for example, calcium chloride, calciumascorbate, calcium gluconate or calcium lactate). In addition,antioxidants and suspending agents can be used.

Lipidoids

The disclosure includes pharmaceutical compositions that comprise alipidoid formulation of the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide.

The synthesis of lipidoids has been extensively described andformulations containing these compounds are particularly suited fordelivery of polynucleotides (see Mahon et al., Bioconjug. Chem. 201021:1448-1454; Schroeder et al., J Intern Med. 2010 267:9-21; Akinc etal., Nat Biotechnol. 2008 26:561-569; Love et al., Proc Natl Acad SciUSA. 2010 107:1864-1869; Siegwart et al., Proc Natl Acad Sci USA. 2011108:12996-3001; all of which are incorporated herein in theirentireties).

Lipidoids have been used to effectively deliver double stranded smallinterfering RNA molecules in rodents and non-human primates (see Akincet al., Nat Biotechnol. 2008 26:561-569; Frank-Kamenetsky et al., ProcNatl Acad Sci USA. 2008 105:11915-11920; Akinc et al., Mol Ther. 200917:872-879; Love et al., Proc Natl Acad Sci USA. 2010 107:1864-1869;Leuschner et al., Nat Biotechnol. 2011 29:1005-1010; all of which isincorporated herein in their entirety), and the present disclosuredescribes their formulation and use in delivering polynucleotides.

Complexes, micelles, liposomes or particles can be prepared containingthese lipidoids and therefore, can result in an effective delivery ofthe polynucleotide, as judged by the production of an encoded protein,following the injection of a lipidoid formulation via localized and/orsystemic routes of administration. Lipidoid complexes of polynucleotidescan be administered by various means including, but not limited to,intravenous, intramuscular, or subcutaneous routes.

In vivo delivery of nucleic acids can be affected by many parameters,including, but not limited to, the formulation composition, nature ofparticle PEGylation, degree of loading, polynucleotide to lipid ratio,and biophysical parameters such as, but not limited to, particle size(Akinc et al., Mol Ther. 2009 17:872-879; herein incorporated byreference in its entirety). As an example, small changes in the anchorchain length of poly(ethylene glycol) (PEG) lipids can result insignificant effects on in vivo efficacy. Formulations with the differentlipidoids, including, but not limited topenta[3-(1-laurylaminopropionyl)]-triethylenetetramine hydrochloride(TETA-5LAP; aka 98N12-5, see Murugaiah et al., Analytical Biochemistry,401:61 (2010); herein incorporated by reference in its entirety),C12-200 (including derivatives and variants), and MD1, can be tested forin vivo activity.

The lipidoid referred to herein as “98N12-5” is disclosed by Akinc etal., Mol Ther. 2009 17:872-879 and is incorporated by reference in itsentirety.

The lipidoid referred to herein as “C12-200” is disclosed by Love etal., Proc Natl Acad Sci USA. 2010 107:1864-1869 and Liu and Huang,Molecular Therapy. 2010 669-670; both of which are herein incorporatedby reference in their entirety. The lipidoid formulations can includeparticles comprising either 3 or 4 or more components in addition topolynucleotides.

Lipidoids and polynucleotide formulations comprising lipidoids aredescribed in International Patent Application No. PCT/US2014/097077, thecontents of which are herein incorporated by reference in its entirety.

Liposomes, Lipoplexes, and Lipid Nanoparticles

The disclosure also includes pharmaceutical compositions that comprise aformulation of the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide, using oneor more liposomes, lipoplexes, or lipid nanoparticles. In oneembodiment, pharmaceutical compositions of the polynucleotides includeliposomes. Liposomes are artificially-prepared vesicles that canprimarily be composed of a lipid bilayer and can be used as a deliveryvehicle for the administration of nutrients and pharmaceuticalformulations. Liposomes can be of different sizes such as, but notlimited to, a multilamellar vesicle (MLV) that can be hundreds ofnanometers in diameter and can contain a series of concentric bilayersseparated by narrow aqueous compartments, a small unicellular vesicle(SUV) that can be smaller than 50 nm in diameter, and a largeunilamellar vesicle (LUV) that can be between 50 and 500 nm in diameter.Liposome design can include, but is not limited to, opsonins or ligandsin order to improve the attachment of liposomes to unhealthy tissue orto activate events such as, but not limited to, endocytosis. Liposomescan contain a low or a high pH in order to improve the delivery of thepharmaceutical formulations.

The formation of liposomes can depend on the physicochemicalcharacteristics such as, but not limited to, the pharmaceuticalformulation entrapped and the liposomal ingredients, the nature of themedium in which the lipid vesicles are dispersed, the effectiveconcentration of the entrapped substance and its potential toxicity, anyadditional processes involved during the application and/or delivery ofthe vesicles, the optimization size, polydispersity and the shelf-lifeof the vesicles for the intended application, and the batch-to-batchreproducibility and possibility of large-scale production of safe andefficient liposomal products.

As a non-limiting example, liposomes such as synthetic membrane vesiclescan be prepared by the methods, apparatus and devices described in USPatent Publication No. US20130177638, US20130177637, US20130177636,US20130177635, US20130177634, US20130177633, US20130183375,US20130183373 and US20130183372, the contents of each of which areherein incorporated by reference in its entirety.

In one embodiment, pharmaceutical compositions described herein caninclude, without limitation, liposomes such as those formed from1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2liposomes from Marina Biotech (Bothell, Wash.),1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA),and MC3 (US20100324120; herein incorporated by reference in itsentirety) and liposomes that can deliver small molecule drugs such as,but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.).

In one embodiment, pharmaceutical compositions described herein caninclude, without limitation, liposomes such as those formed from thesynthesis of stabilized plasmid-lipid particles (SPLP) or stabilizednucleic acid lipid particle (SNALP) that have been previously describedand shown to be suitable for oligonucleotide delivery in vitro and invivo (see Wheeler et al. Gene Therapy. 1999 6:271-281; Zhang et al. GeneTherapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372;Morrissey et al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al.,Nature. 2006 441:111-114; Heyes et al. J Contr Rel. 2005 107:276-287;Semple et al. Nature Biotech. 2010 28:172-176; Judge et al. J ClinInvest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008 19:125-132;U.S. Patent Publication No US20130122104; all of which are incorporatedherein in their entireties). The original manufacture method by Wheeleret al. was a detergent dialysis method, which was later improved byJeffs et al. and is referred to as the spontaneous vesicle formationmethod. The liposome formulations are composed of 3 to 4 lipidcomponents in addition to the polynucleotide. As an example a liposomecan contain, but is not limited to, 55% cholesterol, 20%disteroylphosphatidyl choline (DSPC), 10% PEG-S-DSG, and 15%1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), as described by Jeffset al. As another example, certain liposome formulations can contain,but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30%cationic lipid, where the cationic lipid can be1,2-distearloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA), as described byHeyes et al.

In some embodiments, liposome formulations can comprise from about 25.0%cholesterol to about 40.0% cholesterol, from about 30.0% cholesterol toabout 45.0% cholesterol, from about 35.0% cholesterol to about 50.0%cholesterol and/or from about 48.5% cholesterol to about 60%cholesterol. For example, formulations can comprise a percentage ofcholesterol selected from the group consisting of 28.5%, 31.5%, 33.5%,36.5%, 37.0%, 38.5%, 39.0% and 43.5%. In some embodiments, formulationscan comprise from about 5.0% to about 10.0% DSPC and/or from about 7.0%to about 15.0% DSPC.

In one embodiment, pharmaceutical compositions can include liposomesthat can be formed to deliver polynucleotides that can encode at leastone polypeptide of interest. The polynucleotides can be encapsulated bythe liposome and/or it can be contained in an aqueous core that can thenbe encapsulated by the liposome (see International Pub. Nos.WO2012031046, WO2012031043, WO2012030901 and WO2012006378 and US PatentPublication No. US20130189351, US20130195969 and US20130202684; thecontents of each of which are herein incorporated by reference in theirentirety).

In another embodiment, liposomes can be formulated for targeteddelivery. As a non-limiting example, the liposome can be formulated fortargeted delivery to the liver. The liposome used for targeted deliverycan include, but is not limited to, the liposomes described in andmethods of making liposomes described in US Patent Publication No.US20130195967, the contents of which are herein incorporated byreference in its entirety.

In another embodiment, the polynucleotide can be formulated in acationic oil-in-water emulsion where the emulsion particle comprises anoil core and a cationic lipid that can interact with the polynucleotideanchoring the molecule to the emulsion particle (see International Pub.No. WO2012006380; herein incorporated by reference in its entirety).

In one embodiment, the polynucleotides can be formulated in awater-in-oil emulsion comprising a continuous hydrophobic phase in whichthe hydrophilic phase is dispersed. As a non-limiting example, theemulsion can be made by the methods described in InternationalPublication No. WO201087791, the contents of which are hereinincorporated by reference in its entirety.

In another embodiment, the lipid formulation can include at leastcationic lipid, a lipid that can enhance transfection and a least onelipid that contains a hydrophilic head group linked to a lipid moiety(International Pub. No. WO2011076807 and U.S. Pub. No. 20110200582; thecontents of each of which is herein incorporated by reference in theirentirety). In another embodiment, the polynucleotides can be formulatedin a lipid vesicle that can have crosslinks between functionalized lipidbilayers (see U.S. Pub. No. 20120177724, the contents of which is hereinincorporated by reference in its entirety).

In one embodiment, the polynucleotides can be formulated in a liposomeas described in International Patent Publication No. WO2013086526, thecontents of which is herein incorporated by reference in its entirety.The polynucleotides can be encapsulated in a liposome using reverse pHgradients and/or optimized internal buffer compositions as described inInternational Patent Publication No. WO2013086526, the contents of whichis herein incorporated by reference in its entirety.

In one embodiment, the polynucleotides pharmaceutical compositions canbe formulated in liposomes such as, but not limited to, DiLa2 liposomes(Marina Biotech, Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell,Wash.), neutral DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) basedliposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. CancerBiology & Therapy 2006 5(12)1708-1713); herein incorporated by referencein its entirety) and hyaluronan-coated liposomes (Quiet Therapeutics,Israel).

In one embodiment, the cationic lipid can be a low molecular weightcationic lipid such as those described in US Patent Application No.20130090372, the contents of which are herein incorporated by referencein its entirety.

In one embodiment, the polynucleotides can be formulated in a lipidvesicle that can have crosslinks between functionalized lipid bilayers.

In one embodiment, the polynucleotides can be formulated in a liposomecomprising a cationic lipid. The liposome can have a molar ratio ofnitrogen atoms in the cationic lipid to the phosphates in the RNA (N:Pratio) of between 1:1 and 20:1 as described in International PublicationNo. WO2013006825, herein incorporated by reference in its entirety. Inanother embodiment, the liposome can have a N:P ratio of greater than20:1 or less than 1:1.

In one embodiment, the polynucleotides can be formulated in alipid-polycation complex. The formation of the lipid-polycation complexcan be accomplished by methods known in the art and/or as described inU.S. Pub. No. 20120178702, herein incorporated by reference in itsentirety. As a non-limiting example, the polycation can include acationic peptide or a polypeptide such as, but not limited to,polylysine, polyornithine and/or polyarginine and the cationic peptidesdescribed in International Pub. No. WO2012013326 or US Patent Pub. No.US20130142818; each of which is herein incorporated by reference in itsentirety. In another embodiment, the polynucleotides can be formulatedin a lipid-polycation complex that can further include a non-cationiclipid such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

In one embodiment, the polynucleotides can be formulated in anaminoalcohol lipidoid. Aminoalcohol lipidoids that can be used in thepresent disclosure can be prepared by the methods described in U.S. Pat.No. 8,450,298, herein incorporated by reference in its entirety.

The liposome formulation can be influenced by, but not limited to, theselection of the cationic lipid component, the degree of cationic lipidsaturation, the nature of the PEGylation, ratio of all components andbiophysical parameters such as size. In one example by Semple et al.(Semple et al. Nature Biotech. 2010 28:172-176; herein incorporated byreference in its entirety), the liposome formulation was composed of57.1% cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3%cholesterol, and 1.4% PEG-c-DMA. As another example, changing thecomposition of the cationic lipid could more effectively deliver siRNAto various antigen presenting cells (Basha et al. Mol Ther. 201119:2186-2200; herein incorporated by reference in its entirety). In someembodiments, liposome formulations can comprise from about 35 to about45% cationic lipid, from about 40% to about 50% cationic lipid, fromabout 50% to about 60% cationic lipid and/or from about 55% to about 65%cationic lipid. In some embodiments, the ratio of lipid to mRNA inliposomes can be from about 5:1 to about 20:1, from about 10:1 to about25:1, from about 15:1 to about 30:1 and/or at least 30:1.

In some embodiments, the ratio of PEG in the lipid nanoparticle (LNP)formulations can be increased or decreased and/or the carbon chainlength of the PEG lipid can be modified from C14 to C18 to alter thepharmacokinetics and/or biodistribution of the LNP formulations. As anon-limiting example, LNP formulations can contain from about 0.5% toabout 3.0%, from about 1.0% to about 3.5%, from about 1.5% to about4.0%, from about 2.0% to about 4.5%, from about 2.5% to about 5.0%and/or from about 3.0% to about 6.0% of the lipid molar ratio ofPEG-c-DOMG(R-3-[(co-methoxy-poly(ethyleneglycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine)(also referred to herein as PEG-DOMG) as compared to the cationic lipid,DSPC and cholesterol. In another embodiment the PEG-c-DOMG can bereplaced with a PEG lipid such as, but not limited to, PEG-DSG(1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG(1,2-Dimyristoyl-sn-glycerol) and/or PEG-DPG(1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationiclipid can be selected from any lipid known in the art such as, but notlimited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2-DMA.

In one embodiment, the polynucleotides can be formulated in a lipidnanoparticle such as those described in International Publication No.WO2012170930, the contents of which is herein incorporated by referencein its entirety.

In one embodiment, the formulation comprising the polynucleotide is ananoparticle that can comprise at least one lipid. The lipid can beselected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5,C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG,PEGylated lipids and amino alcohol lipids. In another aspect, the lipidcan be a cationic lipid such as, but not limited to, DLin-DMA,DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA and amino alcohol lipids.The amino alcohol cationic lipid can be the lipids described in and/ormade by the methods described in US Patent Publication No.US20130150625, herein incorporated by reference in its entirety. As anon-limiting example, the cationic lipid can be2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol(Compound 1 in US20130150625);2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol(Compound 2 in US20130150625);2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol(Compound 3 in US20130150625); and2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol(Compound 4 in US20130150625); or any pharmaceutically acceptable saltor stereoisomer thereof.

Lipid nanoparticle formulations typically comprise a lipid, inparticular, an ionizable cationic lipid, for example,2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), ordi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), and furthercomprise a neutral lipid, a sterol and a molecule capable of reducingparticle aggregation, for example a PEG or PEG-modified lipid.

In one embodiment, the lipid nanoparticle formulation consistsessentially of (i) at least one lipid selected from the group consistingof 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); (ii) aneutral lipid selected from DSPC, DPPC, POPC, DOPE and SM; (iii) asterol, e.g., cholesterol; and (iv) a PEG-lipid, e.g., PEG-DMG orPEG-cDMA, in a molar ratio of about 20-60% cationic lipid: 5-25% neutrallipid: 25-55% sterol; 0.5-15% PEG-lipid.

In one embodiment, the formulation includes from about 25% to about 75%on a molar basis of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., fromabout 35 to about 65%, from about 45 to about 65%, about 60%, about57.5%, about 50% or about 40% on a molar basis.

In one embodiment, the formulation includes from about 0.5% to about 15%on a molar basis of the neutral lipid e.g., from about 3 to about 12%,from about 5 to about 10% or about 15%, about 10%, or about 7.5% on amolar basis. Exemplary neutral lipids include, but are not limited to,DSPC, POPC, DPPC, DOPE and SM. In one embodiment, the formulationincludes from about 5% to about 50% on a molar basis of the sterol(e.g., about 15 to about 45%, about 20 to about 40%, about 40%, about38.5%, about 35%, or about 31% on a molar basis. An exemplary sterol ischolesterol. In one embodiment, the formulation includes from about 0.5%to about 20% on a molar basis of the PEG or PEG-modified lipid (e.g.,about 0.5 to about 10%, about 0.5 to about 5%, about 1.5%, about 0.5%,about 1.5%, about 3.5%, or about 5% on a molar basis. In one embodiment,the PEG or PEG modified lipid comprises a PEG molecule of an averagemolecular weight of 2,000 Da. In other embodiments, the PEG or PEGmodified lipid comprises a PEG molecule of an average molecular weightof less than 2,000, for example around 1,500 Da, around 1,000 Da, oraround 500 Da. Exemplary PEG-modified lipids include, but are notlimited to, PEG-distearoyl glycerol (PEG-DMG) (also referred herein asPEG-C14 or C14-PEG), PEG-cDMA (further discussed in Reyes et al. J.Controlled Release, 107, 276-287 (2005) the contents of which are hereinincorporated by reference in its entirety)

In one embodiment, the formulations of the disclosure include 25-75% ofa cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 0.5-15% ofthe neutral lipid, 5-50% of the sterol, and 0.5-20% of the PEG orPEG-modified lipid on a molar basis.

In one embodiment, the formulations of the disclosure include 35-65% ofa cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3-12% of theneutral lipid, 15-45% of the sterol, and 0.5-10% of the PEG orPEG-modified lipid on a molar basis.

In one embodiment, the formulations of the disclosure include 45-65% ofa cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 5-10% of theneutral lipid, 25-40% of the sterol, and 0.5-10% of the PEG orPEG-modified lipid on a molar basis.

In one embodiment, the formulations of the disclosure include about 60%of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 7.5%of the neutral lipid, about 31% of the sterol, and about 1.5% of the PEGor PEG-modified lipid on a molar basis.

In one embodiment, the formulations of the disclosure include about 50%of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% ofthe neutral lipid, about 38.5% of the sterol, and about 1.5% of the PEGor PEG-modified lipid on a molar basis.

In one embodiment, the formulations of the disclosure include about 50%of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% ofthe neutral lipid, about 35% of the sterol, about 4.5% or about 5% ofthe PEG or PEG-modified lipid, and about 0.5% of the targeting lipid ona molar basis.

In one embodiment, the formulations of the disclosure include about 40%of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 15% ofthe neutral lipid, about 40% of the sterol, and about 5% of the PEG orPEG-modified lipid on a molar basis.

In one embodiment, the formulations of the disclosure include about57.2% of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 7.1%of the neutral lipid, about 34.3% of the sterol, and about 1.4% of thePEG or PEG-modified lipid on a molar basis.

In one embodiment, the formulations of the disclosure include about57.5% of a cationic lipid selected from the PEG lipid is PEG-cDMA(PEG-cDMA is further discussed in Reyes et al. (J. Controlled Release,107, 276-287 (2005), the contents of which are herein incorporated byreference in its entirety), about 7.5% of the neutral lipid, about 31.5%of the sterol, and about 3.5% of the PEG or PEG-modified lipid on amolar basis.

In some embodiments, lipid nanoparticle formulation consists essentiallyof a lipid mixture in molar ratios of about 20-70% cationic lipid: 5-45%neutral lipid: 20-55% cholesterol: 0.5-15% PEG-modified lipid. In someembodiments, lipid nanoparticle formulation consists essentially of alipid mixture in molar ratios of about 20-60% cationic lipid: 5-25%neutral lipid: 25-55% cholesterol: 0.5-15% PEG-modified lipid.

In particular embodiments, the molar lipid ratio is approximately50/10/38.5/1.5 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG, PEG-DSG or PEG-DPG),57.2/7.1134.3/1.4 (mol % cationic lipid/neutral lipid, e.g.,DPPC/Chol/PEG-modified lipid, e.g., PEG-cDMA), 40/15/40/5 (mol %cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,PEG-DMG), 50/10/35/4.5/0.5 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DSG), 50/10/35/5 (cationiclipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG),40/10/40/10 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA), 35/15/40/10(mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid,e.g., PEG-DMG or PEG-cDMA) or 52/13/30/5 (mol % cationic lipid/neutrallipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA).

Exemplary lipid nanoparticle compositions and methods of making same aredescribed, for example, in Semple et al. (2010) Nat. Biotechnol.28:172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed., 51:8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578 (thecontents of each of which are incorporated herein by reference in theirentirety).

In one embodiment, the lipid nanoparticle formulations described hereincan comprise a cationic lipid, a PEG lipid and a structural lipid andoptionally comprise a non-cationic lipid. As a non-limiting example, thelipid nanoparticle can comprise about 40-60% of cationic lipid, about5-15% of a non-cationic lipid, about 1-2% of a PEG lipid and about30-50% of a structural lipid. As another non-limiting example, the lipidnanoparticle can comprise about 50% cationic lipid, about 10%non-cationic lipid, about 1.5% PEG lipid and about 38.5% structurallipid. As yet another non-limiting example, the lipid nanoparticle cancomprise about 55% cationic lipid, about 10% non-cationic lipid, about2.5% PEG lipid and about 32.5% structural lipid. In one embodiment, thecationic lipid can be any cationic lipid described herein such as, butnot limited to, DLin-KC2-DMA, DLin-MC3-DMA and L319.

In one embodiment, the lipid nanoparticle formulations described hereincan be 4 component lipid nanoparticles. The lipid nanoparticle cancomprise a cationic lipid, a non-cationic lipid, a PEG lipid and astructural lipid. As a non-limiting example, the lipid nanoparticle cancomprise about 40-60% of cationic lipid, about 5-15% of a non-cationiclipid, about 1-2% of a PEG lipid and about 30-50% of a structural lipid.As another non-limiting example, the lipid nanoparticle can compriseabout 50% cationic lipid, about 10% non-cationic lipid, about 1.5% PEGlipid and about 38.5% structural lipid. As yet another non-limitingexample, the lipid nanoparticle can comprise about 55% cationic lipid,about 10% non-cationic lipid, about 2.5% PEG lipid and about 32.5%structural lipid. In one embodiment, the cationic lipid can be anycationic lipid described herein such as, but not limited to,DLin-KC2-DMA, DLin-MC3-DMA and L319.

In one embodiment, the lipid nanoparticle formulations described hereincan comprise a cationic lipid, a non-cationic lipid, a PEG lipid and astructural lipid. As a non-limiting example, the lipid nanoparticlecomprise about 50% of the cationic lipid DLin-KC2-DMA, about 10% of thenon-cationic lipid DSPC, about 1.5% of the PEG lipid PEG-DOMG and about38.5% of the structural lipid cholesterol. As a non-limiting example,the lipid nanoparticle comprise about 50% of the cationic lipidDLin-MC3-DMA, about 10% of the non-cationic lipid DSPC, about 1.5% ofthe PEG lipid PEG-DOMG and about 38.5% of the structural lipidcholesterol. As a non-limiting example, the lipid nanoparticle compriseabout 50% of the cationic lipid DLin-MC3-DMA, about 10% of thenon-cationic lipid DSPC, about 1.5% of the PEG lipid PEG-DMG and about38.5% of the structural lipid cholesterol. As yet another non-limitingexample, the lipid nanoparticle comprise about 55% of the cationic lipidL319, about 10% of the non-cationic lipid DSPC, about 2.5% of the PEGlipid PEG-DMG and about 32.5% of the structural lipid cholesterol.

In one embodiment, the cationic lipid can be selected from, but notlimited to, a cationic lipid described in International Publication Nos.WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913,WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724,WO201021865, WO2008103276, WO2013086373 and WO2013086354, U.S. Pat. Nos.7,893,302, 7,404,969, 8,283,333, and 8,466,122 and US Patent PublicationNo. US20100036115, US20120202871, US20130064894, US20130129785,US20130150625, US20130178541 and US20130225836; the contents of each ofwhich are herein incorporated by reference in their entirety. In anotherembodiment, the cationic lipid can be selected from, but not limited to,formula A described in International Publication Nos. WO2012040184,WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460,WO2012061259, WO2012054365, WO2012044638 and WO2013116126 or US PatentPublication No. US20130178541 and US20130225836; the contents of each ofwhich is herein incorporated by reference in their entirety. In yetanother embodiment, the cationic lipid can be selected from, but notlimited to, formula CLI-CLXXIX of International Publication No.WO2008103276, formula CLI-CLXXIX of U.S. Pat. No. 7,893,302, formulaCLI-CLXXXXII of U.S. Pat. No. 7,404,969 and formula I-VI of US PatentPublication No. US20100036115, formula I of US Patent Publication NoUS20130123338; each of which is herein incorporated by reference intheir entirety. As a non-limiting example, the cationic lipid can beselected from (20Z,23Z)—N,N-dimethylnonacosa-20,23-dien-10-amine,(17Z,20Z)—N,N-dimemylhexacosa-17,20-dien-9-amine,(1Z,19Z)—N5N-dimethylpentacosa-1 6, 19-dien-8-amine,(13Z,16Z)—N,N-dimethyldocosa-13,16-dien-5-amine,(12Z,15Z)—N,N-dimethylhenicosa-12,15-dien-4-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-6-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-7-amine,(18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-10-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-5-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-4-amine,(19Z,22Z)—N,N-dimeihyloctacosa-19,22-dien-9-amine, (18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-8-amine,(17Z,20Z)—N,N-dimethylhexacosa-17,20-dien-7-amine,(16Z,19Z)—N,N-dimethylpentacosa-16,19-dien-6-amine,(22Z,25Z)—N,N-dimethylhentriaconta-22,25-dien-10-amine, (21Z,24Z)—N,N-dimethyltriaconta-21,24-dien-9-amine,(18Z)—N,N-dimetylheptacos-18-en-10-amine,(17Z)—N,N-dimethylhexacos-17-en-9-amine,(19Z,22Z)—N,N-dimethyloctacosa-19,22-dien-7-amine,N,N-dimethylheptacosan-10-amine,(20Z,23Z)—N-ethyl-N-methylnonacosa-20,23-dien-10-amine,1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl] pyrrolidine,(20Z)—N,N-dimethylheptacos-20-en-10-amine, (15Z)—N,N-dimethyleptacos-15-en-10-amine, (14Z)—N,N-dimethylnonacos-14-en-10-amine,(17Z)—N,N-dimethylnonacos-17-en-10-amine,(24Z)—N,N-dimethyltritriacont-24-en-10-amine,(20Z)—N,N-dimethylnonacos-20-en-10-amine,(22Z)—N,N-dimethylhentriacont-22-en-10-amine,(16Z)—N,N-dimethylpentacos-16-en-8-amine,(12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine,(13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl] eptadecan-8-amine,1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine,N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine,N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine,N,N-dimethyl-1-[(1 S,2R)-2-octylcyclopropyl]hexadecan-8-amine,N,N-dimethyl-[(1R,2 S)-2-undecyIcyclopropyl]tetradecan-5-amine,N,N-dimethyl-3-{7-[(1 S,2R)-2-octylcyclopropyl]heptyl} dodecan-1-amine,1-[(1R,2S)-2-hepty lcyclopropyl]-N,N-dimethyloctadecan-9-amine, 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine,N,N-dimethyl-1-[(1 S,2R)-2-octylcyclopropyl]pentadecan-8-amine,R—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,S—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine,(2S)—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine,(2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,(2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-amine;(2S)—N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine,(2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine,(2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine,1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine,(2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine,1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2R)—N,N-dimethyl-H(1-metoyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,(2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine,N,N-dimethyl-1-{[8-(2-oclylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amineand (11E,20Z,23Z)—N,N-dimethylnonacosa-l1,20,2-trien-10-amine or apharmaceutically acceptable salt or stereoisomer thereof.

In one embodiment, the lipid can be a cleavable lipid such as thosedescribed in International Publication No. WO2012170889, hereinincorporated by reference in its entirety.

In another embodiment, the lipid can be a cationic lipid such as, butnot limited to, Formula (I) of U.S. Patent Application No.US20130064894, the contents of which are herein incorporated byreference in its entirety.

In one embodiment, the cationic lipid can be synthesized by methodsknown in the art and/or as described in International Publication Nos.WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913,WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724,WO201021865, WO2013086373 and WO2013086354; the contents of each ofwhich are herein incorporated by reference in their entirety.

In another embodiment, the cationic lipid can be a trialkyl cationiclipid. Non-limiting examples of trialkyl cationic lipids and methods ofmaking and using the trialkyl cationic lipids are described inInternational Patent Publication No. WO2013126803, the contents of whichare herein incorporated by reference in its entirety.

In one embodiment, the LNP formulations of the polynucleotides cancontain PEG-c-DOMG at 3% lipid molar ratio. In another embodiment, theLNP formulations of polynucleotides can contain PEG-c-DOMG at 1.5% lipidmolar ratio.

In one embodiment, the pharmaceutical compositions of thepolynucleotides can include at least one of the PEGylated lipidsdescribed in International Publication No. WO2012099755, the contents ofwhich is herein incorporated by reference in its entirety.

In one embodiment, the LNP formulation can contain PEG-DMG 2000(1,2-dimyristoyl-sn-glycero-3-phophoethanolamine-N-[methoxy(polyethyleneglycol)-2000). In one embodiment, the LNP formulation can containPEG-DMG 2000, a cationic lipid known in the art and at least one othercomponent. In another embodiment, the LNP formulation can containPEG-DMG 2000, a cationic lipid known in the art, DSPC and cholesterol.As a non-limiting example, the LNP formulation can contain PEG-DMG 2000,DLin-DMA, DSPC and cholesterol. As another non-limiting example the LNPformulation can contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol ina molar ratio of 2:40:10:48 (see, e.g., Geall et al., Nonviral deliveryof self-amplifying RNA vaccines, PNAS 2012; PMID: 22908294; hereinincorporated by reference in its entirety).

In one embodiment, the LNP formulation can be formulated by the methodsdescribed in International Publication Nos. WO2011127255 orWO2008103276, the contents of each of which is herein incorporated byreference in their entirety. As a non-limiting example, thepolynucleotides described herein can be encapsulated in LNP formulationsas described in WO2011127255 and/or WO2008103276; each of which isherein incorporated by reference in their entirety.

In one embodiment, the polynucleotides described herein can beformulated in a nanoparticle to be delivered by a parenteral route asdescribed in U.S. Pub. No. US20120207845; the contents of which areherein incorporated by reference in its entirety.

In one embodiment, the Polynucleotides can be formulated in a lipidnanoparticle made by the methods described in US Patent Publication NoUS20130156845 or International Publication No WO2013093648 orWO2012024526, each of which is herein incorporated by reference in itsentirety.

The lipid nanoparticles described herein can be made in a sterileenvironment by the system and/or methods described in US PatentPublication No. US20130164400, herein incorporated by reference in itsentirety.

In one embodiment, the LNP formulation can be formulated in ananoparticle such as a nucleic acid-lipid particle described in U.S.Pat. No. 8,492,359, the contents of which are herein incorporated byreference in its entirety. As a non-limiting example, the lipid particlecan comprise one or more active agents or therapeutic agents; one ormore cationic lipids comprising from about 50 mol % to about 85 mol % ofthe total lipid present in the particle; one or more non-cationic lipidscomprising from about 13 mol % to about 49.5 mol % of the total lipidpresent in the particle; and one or more conjugated lipids that inhibitaggregation of particles comprising from about 0.5 mol % to about 2 mol% of the total lipid present in the particle. The nucleic acid in thenanoparticle can be the polynucleotides described herein and/or areknown in the art.

In one embodiment, the LNP formulation can be formulated by the methodsdescribed in International Publication Nos. WO2011127255 orWO2008103276, the contents of each of which are herein incorporated byreference in their entirety. As a non-limiting example, modified RNAdescribed herein can be encapsulated in LNP formulations as described inWO2011127255 and/or WO2008103276; the contents of each of which areherein incorporated by reference in their entirety.

In one embodiment, LNP formulations described herein can comprise apolycationic composition. As a non-limiting example, the polycationiccomposition can be selected from formula 1-60 of US Patent PublicationNo. US20050222064; the content of which is herein incorporated byreference in its entirety. In another embodiment, the LNP formulationscomprising a polycationic composition can be used for the delivery ofthe modified RNA described herein in vivo and/or in vitro.

In one embodiment, the LNP formulations described herein canadditionally comprise a permeability enhancer molecule. Non-limitingpermeability enhancer molecules are described in US Patent PublicationNo. US20050222064; the content of which is herein incorporated byreference in its entirety.

In one embodiment, the polynucleotide pharmaceutical compositions can beformulated in liposomes such as, but not limited to, DiLa2 liposomes(Marina Biotech, Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell,Wash.), neutral DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) basedliposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. CancerBiology & Therapy 2006 5(12)1708-1713); herein incorporated by referencein its entirety) and hyaluronan-coated liposomes (Quiet Therapeutics,Israel).

In one embodiment, the Polynucleotides can be formulated in alyophilized gel-phase liposomal composition as described in USPublication No. US2012060293, herein incorporated by reference in itsentirety.

The nanoparticle formulations can comprise a phosphate conjugate. Thephosphate conjugate can increase in vivo circulation times and/orincrease the targeted delivery of the nanoparticle. Phosphate conjugatesfor use with the present disclosure can be made by the methods describedin International Application No. WO2013033438 or US Patent PublicationNo. US20130196948, the contents of each of which are herein incorporatedby reference in its entirety. As a non-limiting example, the phosphateconjugates can include a compound of any one of the formulas describedin International Application No. WO2013033438, herein incorporated byreference in its entirety.

The nanoparticle formulation can comprise a polymer conjugate. Thepolymer conjugate can be a water soluble conjugate. The polymerconjugate can have a structure as described in U.S. Patent ApplicationNo. 20130059360, the contents of which are herein incorporated byreference in its entirety. In one aspect, polymer conjugates with thepolynucleotides of the present disclosure can be made using the methodsand/or segmented polymeric reagents described in U.S. Patent ApplicationNo. 20130072709, herein incorporated by reference in its entirety. Inanother aspect, the polymer conjugate can have pendant side groupscomprising ring moieties such as, but not limited to, the polymerconjugates described in US Patent Publication No. US20130196948, thecontents of which is herein incorporated by reference in its entirety.

The nanoparticle formulations can comprise a conjugate to enhance thedelivery of nanoparticles of the present disclosure in a subject.Further, the conjugate can inhibit phagocytic clearance of thenanoparticles in a subject. In one aspect, the conjugate can be a “self”peptide designed from the human membrane protein CD47 (e.g., the “self”particles described by Rodriguez et al (Science 2013 339, 971-975),herein incorporated by reference in its entirety). As shown by Rodriguezet al. the self peptides delayed macrophage-mediated clearance ofnanoparticles which enhanced delivery of the nanoparticles. In anotheraspect, the conjugate can be the membrane protein CD47 (e.g., seeRodriguez et al. Science 2013 339, 971-975, herein incorporated byreference in its entirety). Rodriguez et al. showed that, similarly to“self” peptides, CD47 can increase the circulating particle ratio in asubject as compared to scrambled peptides and PEG coated nanoparticles.

In one embodiment, the Polynucleotides of the present disclosure areformulated in nanoparticles that comprise a conjugate to enhance thedelivery of the nanoparticles of the present disclosure in a subject.The conjugate can be the CD47 membrane or the conjugate can be derivedfrom the CD47 membrane protein, such as the “self” peptide describedpreviously. In another aspect the nanoparticle can comprise PEG and aconjugate of CD47 or a derivative thereof. In yet another aspect, thenanoparticle can comprise both the “self” peptide described above andthe membrane protein CD47.

In another aspect, a “self” peptide and/or CD47 protein can beconjugated to a virus-like particle or pseudovirion, as described hereinfor delivery of the Polynucleotides of the present disclosure.

In another embodiment, pharmaceutical compositions comprise thepolynucleotides of the present disclosure and a conjugate that can havea degradable linkage. Non-limiting examples of conjugates include anaromatic moiety comprising an ionizable hydrogen atom, a spacer moiety,and a water-soluble polymer. As a non-limiting example, pharmaceuticalcompositions comprising a conjugate with a degradable linkage andmethods for delivering such pharmaceutical compositions are described inUS Patent Publication No. US20130184443, the contents of which areherein incorporated by reference in its entirety.

The nanoparticle formulations can be a carbohydrate nanoparticlecomprising a carbohydrate carrier and a polynucleotide. As anon-limiting example, the carbohydrate carrier can include, but is notlimited to, an anhydride-modified phytoglycogen or glycogen-typematerial, phytoglycogen octenyl succinate, phytoglycogen beta-dextrin,anhydride-modified phytoglycogen beta-dextrin. (See, e.g., InternationalPublication No. WO2012109121; the contents of which are hereinincorporated by reference in its entirety).

Nanoparticle formulations of the present disclosure can be coated with asurfactant or polymer in order to improve the delivery of the particle.In one embodiment, the nanoparticle can be coated with a hydrophiliccoating such as, but not limited to, PEG coatings and/or coatings thathave a neutral surface charge. The hydrophilic coatings can help todeliver nanoparticles with larger payloads such as, but not limited to,Polynucleotides within the central nervous system. As a non-limitingexample nanoparticles comprising a hydrophilic coating and methods ofmaking such nanoparticles are described in US Patent Publication No.US20130183244, the contents of which are herein incorporated byreference in its entirety.

In one embodiment, the lipid nanoparticles of the present disclosure canbe hydrophilic polymer particles. Non-limiting examples of hydrophilicpolymer particles and methods of making hydrophilic polymer particlesare described in US Patent Publication No. US20130210991, the contentsof which are herein incorporated by reference in its entirety.

In another embodiment, the lipid nanoparticles of the present disclosurecan be hydrophobic polymer particles.

Lipid nanoparticle formulations can be improved by replacing thecationic lipid with a biodegradable cationic lipid that is known as arapidly eliminated lipid nanoparticle (reLNP). Ionizable cationiclipids, such as, but not limited to, DLinDMA, DLin-KC2-DMA, andDLin-MC3-DMA, have been shown to accumulate in plasma and tissues overtime and can be a potential source of toxicity. The rapid metabolism ofthe rapidly eliminated lipids can improve the tolerability andtherapeutic index of the lipid nanoparticles by an order of magnitudefrom a 1 mg/kg dose to a 10 mg/kg dose in rat. Inclusion of anenzymatically degraded ester linkage can improve the degradation andmetabolism profile of the cationic component, while still maintainingthe activity of the reLNP formulation. The ester linkage can beinternally located within the lipid chain or it can be terminallylocated at the terminal end of the lipid chain. The internal esterlinkage can replace any carbon in the lipid chain.

In one embodiment, the internal ester linkage can be located on eitherside of the saturated carbon.

Lipid nanoparticles can be engineered to alter the surface properties ofparticles so the lipid nanoparticles can penetrate the mucosal barrier.Mucus is located on mucosal tissue such as, but not limited to, oral(e.g., the buccal and esophageal membranes and tonsil tissue),ophthalmic, gastrointestinal (e.g., stomach, small intestine, largeintestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal,tracheal and bronchial membranes), genital (e.g., vaginal, cervical andurethral membranes). Nanoparticles larger than 10-200 nm can be used forhigher drug encapsulation efficiency and the ability to provide thesustained delivery of a wide array of drugs have been thought to be toolarge to rapidly diffuse through mucosal barriers. Mucus is continuouslysecreted, shed, discarded or digested and recycled so most of thetrapped particles can be removed from the mucosal tissue within secondsor within a few hours. Large polymeric nanoparticles (200 nm-500 nm indiameter) that have been coated densely with a low molecular weightpolyethylene glycol (PEG) diffused through mucus only 4 to 6-fold lowerthan the same particles diffusing in water (Lai et al. PNAS 2007104(5):1482-487; Lai et al. Adv Drug Deliv Rev. 2009 61(2): 158-171;each of which is herein incorporated by reference in their entirety).The transport of nanoparticles can be determined using rates ofpermeation and/or fluorescent microscopy techniques including, but notlimited to, fluorescence recovery after photobleaching (FRAP) and highresolution multiple particle tracking (MPT). As a non-limiting example,compositions that can penetrate a mucosal barrier can be made asdescribed in U.S. Pat. No. 8,241,670 or International Patent PublicationNo. WO2013110028, the contents of each of which are herein incorporatedby reference in its entirety.

The lipid nanoparticle engineered to penetrate mucus can comprise apolymeric material (i.e., a polymeric core) and/or a polymer-vitaminconjugate and/or a tri-block co-polymer. The polymeric material caninclude, but is not limited to, polyamines, polyethers, polyamides,polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes),polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes,polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates,polyacrylonitriles, and polyarylates. The polymeric material can bebiodegradable and/or biocompatible. Non-limiting examples ofbiocompatible polymers are described in International Patent PublicationNo. WO2013116804, the contents of which are herein incorporated byreference in its entirety. The polymeric material can additionally beirradiated. As a non-limiting example, the polymeric material can begamma irradiated (See e.g., International App. No. WO201282165, hereinincorporated by reference in its entirety). Non-limiting examples ofspecific polymers include poly(caprolactone) (PCL), ethylene vinylacetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid)(PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid)(PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide)(PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone),poly(D,L-lactide-co-caprolactone-co-glycolide),poly(D,L-lactide-co-PEO-co-D,L-lactide),poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate,polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA),polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids),polyanhydrides, polyorthoesters, poly(ester amides), polyamides,poly(ester ethers), polycarbonates, polyalkylenes such as polyethyleneand polypropylene, polyalkylene glycols such as poly(ethylene glycol)(PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such aspoly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinylethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halidessuch as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes,polystyrene (PS), polyurethanes, derivatized celluloses such as alkylcelluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters,nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose,polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA),poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate),poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate),poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate),poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) andcopolymers and mixtures thereof, polydioxanone and its copolymers,polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene,poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid),poly(lactide-co-caprolactone), PEG-PLGA-PEG and trimethylene carbonate,polyvinylpyrrolidone. The lipid nanoparticle can be coated or associatedwith a co-polymer such as, but not limited to, a block co-polymer (suchas a branched polyether-polyamide block copolymer described inInternational Publication No. WO2013012476, herein incorporated byreference in its entirety), and (poly(ethylene glycol))-(poly(propyleneoxide))-(poly(ethylene glycol)) triblock copolymer (see, e.g., USPublication 20120121718 and US Publication 20100003337 and U.S. Pat. No.8,263,665; each of which is herein incorporated by reference in theirentirety). The co-polymer can be a polymer that is generally regarded assafe (GRAS) and the formation of the lipid nanoparticle can be in such away that no new chemical entities are created. For example, the lipidnanoparticle can comprise poloxamers coating PLGA nanoparticles withoutforming new chemical entities that are still able to rapidly penetratehuman mucus (Yang et al. Angew. Chem. Int. Ed. 2011 50:2597-2600; thecontents of which are herein incorporated by reference in its entirety).A non-limiting scalable method to produce nanoparticles that canpenetrate human mucus is described by Xu et al. (See, e.g., J ControlRelease 2013, 170(2):279-86; the contents of which are hereinincorporated by reference in its entirety).

The vitamin of the polymer-vitamin conjugate can be vitamin E. Thevitamin portion of the conjugate can be substituted with other suitablecomponents such as, but not limited to, vitamin A, vitamin E, othervitamins, cholesterol, a hydrophobic moiety, or a hydrophobic componentof other surfactants (e.g., sterol chains, fatty acids, hydrocarbonchains and alkylene oxide chains).

The lipid nanoparticle engineered to penetrate mucus can include surfacealtering agents such as, but not limited to, polynucleotides, anionicproteins (e.g., bovine serum albumin), surfactants (e.g., cationicsurfactants such as for example dimethyldioctadecyl-ammonium bromide),sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids,polymers (e.g., heparin, polyethylene glycol and poloxamer), mucolyticagents (e.g., N-acetylcysteine, mugwort, bromelain, papain,clerodendrum, acetylcysteine, bromhexine, carbocisteine, eprazinone,mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin,gelsolin, thymosin β4 dornase alfa, neltenexine, erdosteine) and variousDNases including rhDNase. The surface altering agent can be embedded orenmeshed in the particle's surface or disposed (e.g., by coating,adsorption, covalent linkage, or other process) on the surface of thelipid nanoparticle. (see e.g., US Publication 20100215580 and USPublication 20080166414 and US20130164343; the contents of each of whichis herein incorporated by reference in their entirety).

In one embodiment, the mucus penetrating lipid nanoparticles cancomprise at least one polynucleotide described herein. Thepolynucleotide can be encapsulated in the lipid nanoparticle and/ordisposed on the surface of the particle. The polynucleotide can becovalently coupled to the lipid nanoparticle. Formulations of mucuspenetrating lipid nanoparticles can comprise a plurality ofnanoparticles. Further, the formulations can contain particles that caninteract with the mucus and alter the structural and/or adhesiveproperties of the surrounding mucus to decrease mucoadhesion that canincrease the delivery of the mucus penetrating lipid nanoparticles tothe mucosal tissue.

In another embodiment, the mucus penetrating lipid nanoparticles can bea hypotonic formulation comprising a mucosal penetration enhancingcoating. The formulation can be hypotonic for the epithelium to which itis being delivered. Non-limiting examples of hypotonic formulations canbe found in International Patent Publication No. WO2013110028, thecontents of which are herein incorporated by reference in its entirety.

In one embodiment, in order to enhance the delivery through the mucosalbarrier the polynucleotide formulation can comprise or be a hypotonicsolution. Hypotonic solutions were found to increase the rate at whichmucoinert particles such as, but not limited to, mucus-penetratingparticles, were able to reach the vaginal epithelial surface (see, e.g.,Ensign et al. Biomaterials 2013 34(28):6922-9; the contents of which isherein incorporated by reference in its entirety).

In one embodiment, the polynucleotide is formulated as a lipoplex, suchas, without limitation, the ATUPLEX™ system, the DACC system, the DBTCsystem and other siRNA-lipoplex technology from Silence Therapeutics(London, United Kingdom), STEMFECT™ from STEMGENT® (Cambridge, Mass.),and polyethylenimine (PEI) or protamine-based targeted and non-targeteddelivery of nucleic acids (Aleku et al. Cancer Res. 2008 68:9788-9798;Strumberg et al. Int J Clin Pharmacol Ther 2012 50:76-78; Santel et al.,Gene Ther 2006 13:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370;Gutbier et al., Pulm Pharmacol. Ther. 2010 23:334-344; Kaufmann et al.Microvasc Res 2010 80:286-293 Weide et al. J Immunother. 200932:498-507; Weide et al. J Immunother. 2008 31:180-188; Pascolo ExpertOpin. Biol. Ther. 4:1285-1294; Fotin-Mleczek et al., 2011 J. Immunother.34:1-15; Song et al., Nature Biotechnol. 2005, 23:709-717; Peer et al.,Proc Natl Acad Sci USA. 2007 6; 104:4095-4100; deFougerolles Hum GeneTher. 2008 19:125-132; all of which are incorporated herein by referencein its entirety).

In one embodiment such formulations can also be constructed orcompositions altered such that they passively or actively are directedto different cell types in vivo, including but not limited tohepatocytes, immune cells, tumor cells, endothelial cells, antigenpresenting cells, and leukocytes (Akinc et al. Mol Ther. 201018:1357-1364; Song et al., Nat Biotechnol. 2005 23:709-717; Judge etal., J Clin Invest. 2009 119:661-673; Kaufmann et al., Microvasc Res2010 80:286-293; Santel et al., Gene Ther 2006 13:1222-1234; Santel etal., Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther.2010 23:334-344; Basha et al., Mol. Ther. 2011 19:2186-2200; Fenske andCullis, Expert Opin Drug Deliv. 2008 5:25-44; Peer et al., Science. 2008319:627-630; Peer and Lieberman, Gene Ther. 2011 18:1127-1133; all ofwhich are incorporated herein by reference in its entirety). One exampleof passive targeting of formulations to liver cells includes theDLin-DMA, DLin-KC2-DMA and DLin-MC3-DMA-based lipid nanoparticleformulations that have been shown to bind to apolipoprotein E andpromote binding and uptake of these formulations into hepatocytes invivo (Akinc et al. Mol Ther. 2010 18:1357-1364; herein incorporated byreference in its entirety). Formulations can also be selectivelytargeted through expression of different ligands on their surface asexemplified by, but not limited by, folate, transferrin,N-acetylgalactosamine (GalNAc), and antibody targeted approaches(Kolhatkar et al., Curr Drug Discov Technol. 2011 8:197-206; Musacchioand Torchilin, Front Biosci. 2011 16:1388-1412; Yu et al., Mol MembrBiol. 2010 27:286-298; Patil et al., Crit Rev Ther Drug Carrier Syst.2008 25:1-61; Benoit et al., Biomacromolecules. 2011 12:2708-2714; Zhaoet al., Expert Opin Drug Deliv. 2008 5:309-319; Akinc et al., Mol Ther.2010 18:1357-1364; Srinivasan et al., Methods Mol Biol. 2012820:105-116; Ben-Arie et al., Methods Mol Biol. 2012 757:497-507; Peer2010 J Control Release. 20:63-68; Peer et al., Proc Natl Acad Sci USA.2007 104:4095-4100; Kim et al., Methods Mol Biol. 2011 721:339-353;Subramanya et al., Mol Ther. 2010 18:2028-2037; Song et al., NatBiotechnol. 2005 23:709-717; Peer et al., Science. 2008 319:627-630;Peer and Lieberman, Gene Ther. 2011 18:1127-1133; all of which areincorporated herein by reference in its entirety).

In one embodiment, the polynucleotide is formulated as a solid lipidnanoparticle. A solid lipid nanoparticle (SLN) can be spherical with anaverage diameter between 10 to 1000 nm. SLN possess a solid lipid corematrix that can solubilize lipophilic molecules and can be stabilizedwith surfactants and/or emulsifiers. In a further embodiment, the lipidnanoparticle can be a self-assembly lipid-polymer nanoparticle (seeZhang et al., ACS Nano, 2008, 2 (8), pp 1696-1702; the contents of whichare herein incorporated by reference in its entirety). As a non-limitingexample, the SLN can be the SLN described in International PatentPublication No. WO2013105101, the contents of which are hereinincorporated by reference in its entirety. As another non-limitingexample, the SLN can be made by the methods or processes described inInternational Patent Publication No. WO2013105101, the contents of whichare herein incorporated by reference in its entirety.

Liposomes, lipoplexes, or lipid nanoparticles can be used to improve theefficacy of polynucleotides directed protein production as theseformulations can be able to increase cell transfection by thepolynucleotide; and/or increase the translation of encoded protein. Onesuch example involves the use of lipid encapsulation to enable theeffective systemic delivery of polyplex plasmid DNA (Heyes et al., MolTher. 2007 15:713-720; herein incorporated by reference in itsentirety). The liposomes, lipoplexes, or lipid nanoparticles can also beused to increase the stability of the polynucleotide.

In one embodiment, the Polynucleotides of the present disclosure can beformulated for controlled release and/or targeted delivery. As usedherein, “controlled release” refers to a pharmaceutical composition orcompound release profile that conforms to a particular pattern ofrelease to effect a therapeutic outcome. In one embodiment, thepolynucleotides can be encapsulated into a delivery agent describedherein and/or known in the art for controlled release and/or targeteddelivery. As used herein, the term “encapsulate” means to enclose,surround or encase. As it relates to the formulation of the compounds ofthe disclosure, encapsulation can be substantial, complete or partial.The term “substantially encapsulated” means that at least greater than50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9 or greater than99.999% of the pharmaceutical composition or compound of the disclosurecan be enclosed, surrounded or encased within the delivery agent.“Partially encapsulation” means that less than 10, 10, 20, 30, 40 50 orless of the pharmaceutical composition or compound of the disclosure canbe enclosed, surrounded or encased within the delivery agent.Advantageously, encapsulation can be determined by measuring the escapeor the activity of the pharmaceutical composition or compound of thedisclosure using fluorescence and/or electron micrograph. For example,at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98,99, 99.9, 99.99 or greater than 99.99% of the pharmaceutical compositionor compound of the disclosure are encapsulated in the delivery agent.

In one embodiment, the controlled release formulation can include, butis not limited to, tri-block co-polymers. As a non-limiting example, theformulation can include two different types of tri-block co-polymers(International Pub. No. WO2012131104 and WO2012131106; the contents ofeach of which is herein incorporated by reference in its entirety).

In another embodiment, the Polynucleotides can be encapsulated into alipid nanoparticle or a rapidly eliminated lipid nanoparticle and thelipid nanoparticles or a rapidly eliminated lipid nanoparticle can thenbe encapsulated into a polymer, hydrogel and/or surgical sealantdescribed herein and/or known in the art. As a non-limiting example, thepolymer, hydrogel or surgical sealant can be PLGA, ethylene vinylacetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua,Fla.), HYLENEX® (Halozyme Therapeutics, San Diego Calif.), surgicalsealants such as fibrinogen polymers (Ethicon Inc. Cornelia, Ga.),TISSELL® (Baxter International, Inc. Deerfield, Ill.), PEG-basedsealants, and COSEAL® (Baxter International, Inc. Deerfield, Ill.).

In another embodiment, the lipid nanoparticle can be encapsulated intoany polymer known in the art that can form a gel when injected into asubject. As another non-limiting example, the lipid nanoparticle can beencapsulated into a polymer matrix that can be biodegradable.

In one embodiment, the polynucleotide formulation for controlled releaseand/or targeted delivery can also include at least one controlledrelease coating. Controlled release coatings include, but are notlimited to, OPADRY®, polyvinylpyrrolidone/vinyl acetate copolymer,polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropylcellulose, hydroxyethyl cellulose, EUDRAGIT RL®, EUDRAGIT RS® andcellulose derivatives such as ethylcellulose aqueous dispersions(AQUACOAT® and SURELEASE®).

In one embodiment, the polynucleotide controlled release and/or targeteddelivery formulation can comprise at least one degradable polyester thatcan contain polycationic side chains. Degradable polyesters include, butare not limited to, poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester), and combinations thereof. In anotherembodiment, the degradable polyesters can include a PEG conjugation toform a PEGylated polymer.

In one embodiment, the polynucleotide controlled release and/or targeteddelivery formulation comprising at least one polynucleotide can compriseat least one PEG and/or PEG related polymer derivatives as described inU.S. Pat. No. 8,404,222, herein incorporated by reference in itsentirety.

In another embodiment, the polynucleotide controlled release deliveryformulation comprising at least one polynucleotide can be the controlledrelease polymer system described in US20130130348, herein incorporatedby reference in its entirety.

In one embodiment, the Polynucleotides of the present disclosure can beencapsulated in a therapeutic nanoparticle, referred to herein as“therapeutic nanoparticle polynucleotides.” Therapeutic nanoparticlescan be formulated by methods described herein and known in the art suchas, but not limited to, International Pub Nos. WO2010005740,WO2010030763, WO2010005721, WO2010005723, WO2012054923, US Pub. Nos.US20110262491, US20100104645, US20100087337, US20100068285,US20110274759, US20100068286, US20120288541, US20130123351 andUS20130230567 and U.S. Pat. Nos. 8,206,747, 8,293,276, 8,318,208 and8,318,211; the contents of each of which are herein incorporated byreference in their entirety. In another embodiment, therapeutic polymernanoparticles can be identified by the methods described in US Pub No.US20120140790, the contents of which is herein incorporated by referencein its entirety.

In one embodiment, the therapeutic nanoparticle polynucleotide can beformulated for sustained release. As used herein, “sustained release”refers to a pharmaceutical composition or compound that conforms to arelease rate over a specific period of time. The period of time caninclude, but is not limited to, hours, days, weeks, months and years. Asa non-limiting example, the sustained release nanoparticle can comprisea polymer and a therapeutic agent such as, but not limited to, thepolynucleotides of the present disclosure (see International Pub No.2010075072 and US Pub No. US20100216804, US20110217377 andUS20120201859, each of which is herein incorporated by reference intheir entirety). In another non-limiting example, the sustained releaseformulation can comprise agents that permit persistent bioavailabilitysuch as, but not limited to, crystals, macromolecular gels and/orparticulate suspensions (see US Patent Publication No US20130150295, thecontents of which is herein incorporated by reference in its entirety).

In one embodiment, the therapeutic nanoparticle Polynucleotides can beformulated to be target specific. As a non-limiting example, thetherapeutic nanoparticles can include a corticosteroid (seeInternational Pub. No. WO2011084518; herein incorporated by reference inits entirety). As a non-limiting example, the therapeutic nanoparticlescan be formulated in nanoparticles described in International Pub No.WO2008121949, WO2010005726, WO2010005725, WO2011084521 and US Pub No.US20100069426, US20120004293 and US20100104655, each of which is hereinincorporated by reference in their entirety.

In one embodiment, the nanoparticles of the present disclosure cancomprise a polymeric matrix. As a non-limiting example, the nanoparticlecan comprise two or more polymers such as, but not limited to,polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids,polypropylfumerates, polycaprolactones, polyamides, polyacetals,polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinylalcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polycyanoacrylates, polyureas, polystyrenes,polyamines, polylysine, poly(ethylene imine), poly(serine ester),poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) orcombinations thereof.

In one embodiment, the therapeutic nanoparticle comprises a diblockcopolymer. In one embodiment, the diblock copolymer can include PEG incombination with a polymer such as, but not limited to, polyethylenes,polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates,polycaprolactones, polyamides, polyacetals, polyethers, polyesters,poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine,poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester) or combinations thereof. In anotherembodiment, the diblock copolymer can comprise the diblock copolymersdescribed in European Patent Publication No. WO2012166923, the contentsof which are herein incorporated by reference in its entirety. In yetanother embodiment, the diblock copolymer can be a high-X diblockcopolymer such as those described in International Patent PublicationNo. WO2013120052, the contents of which are herein incorporated byreference in its entirety.

As a non-limiting example the therapeutic nanoparticle comprises aPLGA-PEG block copolymer (see US Pub. No. US20120004293 and U.S. Pat.No. 8,236,330, each of which is herein incorporated by reference intheir entirety). In another non-limiting example, the therapeuticnanoparticle is a stealth nanoparticle comprising a diblock copolymer ofPEG and PLA or PEG and PLGA (see U.S. Pat. No. 8,246,968 andInternational Publication No. WO2012166923, the contents of each ofwhich are herein incorporated by reference in its entirety). In yetanother non-limiting example, the therapeutic nanoparticle is a stealthnanoparticle or a target-specific stealth nanoparticle as described inUS Patent Publication No. US20130172406, the contents of which areherein incorporated by reference in its entirety.

In one embodiment, the therapeutic nanoparticle can comprise amultiblock copolymer (see, e.g., U.S. Pat. Nos. 8,263,665 and 8,287,910and US Patent Pub. No. US20130195987; the contents of each of which areherein incorporated by reference in its entirety).

In yet another non-limiting example, the lipid nanoparticle comprisesthe block copolymer PEG-PLGA-PEG (see, e.g., the thermosensitivehydrogel (PEG-PLGA-PEG) was used as a TGF-beta1 gene delivery vehicle inLee et al. Thermosensitive Hydrogel as a Tgf-β1 Gene Delivery VehicleEnhances Diabetic Wound Healing. Pharmaceutical Research, 2003 20(12):1995-2000; as a controlled gene delivery system in Li et al. ControlledGene Delivery System Based on Thermosensitive Biodegradable Hydrogel.Pharmaceutical Research 2003 20(6):884-888; and Chang et al., Non-ionicamphiphilic biodegradable PEG-PLGA-PEG copolymer enhances gene deliveryefficiency in rat skeletal muscle. J Controlled Release. 2007118:245-253; each of which is herein incorporated by reference in itsentirety). The Polynucleotides of the present disclosure can beformulated in lipid nanoparticles comprising the PEG-PLGA-PEG blockcopolymer.

In one embodiment, the therapeutic nanoparticle can comprise amultiblock copolymer (see, e.g., U.S. Pat. Nos. 8,263,665 and 8,287,910and US Patent Pub. No. US20130195987; the contents of each of which areherein incorporated by reference in its entirety).

In one embodiment, the block copolymers described herein can be includedin a polyion complex comprising a non-polymeric micelle and the blockcopolymer (see, e.g., U.S. Pub. No. 20120076836; herein incorporated byreference in its entirety).

In one embodiment, the therapeutic nanoparticle can comprise at leastone acrylic polymer. Acrylic polymers include but are not limited to,acrylic acid, methacrylic acid, acrylic acid and methacrylic acidcopolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates,cyanoethyl methacrylate, amino alkyl methacrylate copolymer,poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates andcombinations thereof.

In one embodiment, the therapeutic nanoparticles can comprise at leastone poly(vinyl ester) polymer. The poly(vinyl ester) polymer can be acopolymer such as a random copolymer. As a non-limiting example, therandom copolymer can have a structure such as those described inInternational Application No. WO2013032829 or US Patent Publication NoUS20130121954, the contents of which are herein incorporated byreference in its entirety. In one aspect, the poly(vinyl ester) polymerscan be conjugated to the polynucleotides described herein. In anotheraspect, the poly(vinyl ester) polymer that can be used in the presentdisclosure can be those described in, herein incorporated by referencein its entirety.

In one embodiment, the therapeutic nanoparticle can comprise at leastone diblock copolymer. The diblock copolymer can be, but it not limitedto, a poly(lactic) acid-poly(ethylene)glycol copolymer (see, e.g.,International Patent Publication No. WO2013044219; herein incorporatedby reference in its entirety).

In one embodiment, the therapeutic nanoparticles can comprise at leastone cationic polymer described herein and/or known in the art.

In one embodiment, the therapeutic nanoparticles can comprise at leastone amine-containing polymer such as, but not limited to polylysine,polyethylene imine, poly(amidoamine) dendrimers, poly(beta-amino esters)(see, e.g., U.S. Pat. No. 8,287,849; herein incorporated by reference inits entirety) and combinations thereof.

In another embodiment, the nanoparticles described herein can comprisean amine cationic lipid such as those described in International PatentApplication No. WO2013059496, the contents of which are hereinincorporated by reference in its entirety. In one aspect the cationiclipids can have an amino-amine or an amino-amide moiety.

In one embodiment, the therapeutic nanoparticles can comprise at leastone degradable polyester that can contain polycationic side chains.Degradable polyesters include, but are not limited to, poly(serineester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester),and combinations thereof. In another embodiment, the degradablepolyesters can include a PEG conjugation to form a PEGylated polymer.

In another embodiment, the therapeutic nanoparticle can include aconjugation of at least one targeting ligand. The targeting ligand canbe any ligand known in the art such as, but not limited to, a monoclonalantibody. (Kirpotin et al, Cancer Res. 2006 66:6732-6740; hereinincorporated by reference in its entirety).

In one embodiment, the therapeutic nanoparticle Polynucleotides, e.g.,therapeutic nanoparticles comprising at least one polynucleotide can beformulated using the methods described by Podobinski et al in U.S. Pat.No. 8,404,799, the contents of which are herein incorporated byreference in its entirety.

In one embodiment, the Polynucleotides can be encapsulated in, linked toand/or associated with synthetic nanocarriers. Synthetic nanocarriersinclude, but are not limited to, those described in International Pub.Nos. WO2010005740, WO2010030763, WO201213501, WO2012149252,WO2012149255, WO2012149259, WO2012149265, WO2012149268, WO2012149282,WO2012149301, WO2012149393, WO2012149405, WO2012149411, WO2012149454 andWO2013019669, and US Pub. Nos. US20110262491, US20100104645,US20100087337 and US20120244222, each of which is herein incorporated byreference in their entirety. The synthetic nanocarriers can beformulated using methods known in the art and/or described herein. As anon-limiting example, the synthetic nanocarriers can be formulated bythe methods described in International Pub Nos. WO2010005740,WO2010030763 and WO201213501 and US Pub. Nos. US20110262491,US20100104645, US20100087337 and US2012024422, each of which is hereinincorporated by reference in their entirety. In another embodiment, thesynthetic nanocarrier formulations can be lyophilized by methodsdescribed in International Pub. No. WO2011072218 and U.S. Pat. No.8,211,473; the content of each of which is herein incorporated byreference in their entirety. In yet another embodiment, formulations ofthe present disclosure, including, but not limited to, syntheticnanocarriers, can be lyophilized or reconstituted by the methodsdescribed in US Patent Publication No. US20130230568, the contents ofwhich are herein incorporated by reference in its entirety.

In one embodiment, the synthetic nanocarriers can contain reactivegroups to release the polynucleotides described herein (seeInternational Pub. No. WO20120952552 and US Pub No. US20120171229, eachof which is herein incorporated by reference in their entirety).

In one embodiment, the synthetic nanocarriers can be formulated fortargeted release. In one embodiment, the synthetic nanocarrier isformulated to release the polynucleotides at a specified pH and/or aftera desired time interval. As a non-limiting example, the syntheticnanoparticle can be formulated to release the polynucleotides after 24hours and/or at a pH of 4.5 (see International Pub. Nos. WO2010138193and WO2010138194 and US Pub Nos. US20110020388 and US20110027217, eachof which is herein incorporated by reference in their entireties).

In one embodiment, the synthetic nanocarriers can be formulated forcontrolled and/or sustained release of the polynucleotides describedherein. As a non-limiting example, the synthetic nanocarriers forsustained release can be formulated by methods known in the art,described herein and/or as described in International Pub No.WO2010138192 and US Pub No. 20100303850, each of which is hereinincorporated by reference in their entirety.

In one embodiment, the polynucleotide can be formulated for controlledand/or sustained release wherein the formulation comprises at least onepolymer that is a crystalline side chain (CYSC) polymer. CYSC polymersare described in U.S. Pat. No. 8,399,007, herein incorporated byreference in its entirety.

In one embodiment, the synthetic nanocarrier can comprise at least onepolynucleotide that encodes at least one adjuvant. As non-limitingexample, the adjuvant can comprise dimethyldioctadecylammonium-bromide,dimethyldioctadecylammonium-chloride,dimethyldioctadecylammonium-phosphate ordimethyldioctadecylammonium-acetate (DDA) and an apolar fraction or partof said apolar fraction of a total lipid extract of a mycobacterium(See, e.g., U.S. Pat. No. 8,241,610; herein incorporated by reference inits entirety). In another embodiment, the synthetic nanocarrier cancomprise at least one polynucleotide and an adjuvant. As a non-limitingexample, the synthetic nanocarrier comprising and adjuvant can beformulated by the methods described in International Pub No.WO2011150240 and US Pub No. US20110293700, each of which is hereinincorporated by reference in its entirety.

In one embodiment, the synthetic nanocarrier can encapsulate at leastone polynucleotide that encodes a peptide, fragment or region from avirus. As a non-limiting example, the synthetic nanocarrier can include,but is not limited to, the nanocarriers described in International PubNo. WO2012024621, WO201202629, WO2012024632 and US Pub No.US20120064110, US20120058153 and US20120058154, each of which is hereinincorporated by reference in their entirety.

In one embodiment, the synthetic nanocarrier can be coupled to apolynucleotide that can be able to trigger a humoral and/or cytotoxic Tlymphocyte (CTL) response (see, e.g., International Publication No.WO2013019669, herein incorporated by reference in its entirety).

In one embodiment, the polynucleotide can be encapsulated in, linked toand/or associated with zwitterionic lipids. Non-limiting examples ofzwitterionic lipids and methods of using zwitterionic lipids aredescribed in US Patent Publication No. US20130216607, the contents ofwhich are herein incorporated by reference in its entirety. In oneaspect, the zwitterionic lipids can be used in the liposomes and lipidnanoparticles described herein.

In one embodiment, the polynucleotide can be formulated in colloidnanocarriers as described in US Patent Publication No. US20130197100,the contents of which are herein incorporated by reference in itsentirety.

In one embodiment, the nanoparticle can be optimized for oraladministration. The nanoparticle can comprise at least one cationicbiopolymer such as, but not limited to, chitosan or a derivativethereof. As a non-limiting example, the nanoparticle can be formulatedby the methods described in U.S. Pub. No. 20120282343; hereinincorporated by reference in its entirety.

In some embodiments, LNPs comprise the lipid KL52 (an amino-lipiddisclosed in U.S. Application Publication No. 2012/0295832 expresslyincorporated herein by reference in its entirety). Activity and/orsafety (as measured by examining one or more of ALT/AST, white bloodcell count and cytokine induction) of LNP administration can be improvedby incorporation of such lipids. LNPs comprising KL52 can beadministered intravenously and/or in one or more doses. In someembodiments, administration of LNPs comprising KL52 results in equal orimproved mRNA and/or protein expression as compared to LNPs comprisingMC3.

In some embodiments, polynucleotide can be delivered using smaller LNPs.Such particles can comprise a diameter from below 0.1 um up to 100 nmsuch as, but not limited to, less than 0.1 um, less than 1.0 um, lessthan 5 um, less than 10 um, less than 15 um, less than 20 um, less than25 um, less than 30 um, less than 35 um, less than 40 um, less than 50um, less than 55 um, less than 60 um, less than 65 um, less than 70 um,less than 75 um, less than 80 um, less than 85 um, less than 90 um, lessthan 95 um, less than 100 um, less than 125 um, less than 150 um, lessthan 175 um, less than 200 um, less than 225 um, less than 250 um, lessthan 275 um, less than 300 um, less than 325 um, less than 350 um, lessthan 375 um, less than 400 um, less than 425 um, less than 450 um, lessthan 475 um, less than 500 um, less than 525 um, less than 550 um, lessthan 575 um, less than 600 um, less than 625 um, less than 650 um, lessthan 675 um, less than 700 um, less than 725 um, less than 750 um, lessthan 775 um, less than 800 um, less than 825 um, less than 850 um, lessthan 875 um, less than 900 um, less than 925 um, less than 950 um, orless than 975 um.

In another embodiment, polynucleotides can be delivered using smallerLNPs that can comprise a diameter from about 1 nm to about 100 nm, fromabout 1 nm to about 10 nm, about 1 nm to about 20 nm, from about 1 nm toabout 30 nm, from about 1 nm to about 40 nm, from about 1 nm to about 50nm, from about 1 nm to about 60 nm, from about 1 nm to about 70 nm, fromabout 1 nm to about 80 nm, from about 1 nm to about 90 nm, from about 5nm to about from 100 nm, from about 5 nm to about 10 nm, about 5 nm toabout 20 nm, from about 5 nm to about 30 nm, from about 5 nm to about 40nm, from about 5 nm to about 50 nm, from about 5 nm to about 60 nm, fromabout 5 nm to about 70 nm, from about 5 nm to about 80 nm, from about 5nm to about 90 nm, about 10 to about 50 nM, from about 20 to about 50nm, from about 30 to about 50 nm, from about 40 to about 50 nm, fromabout 20 to about 60 nm, from about 30 to about 60 nm, from about 40 toabout 60 nm, from about 20 to about 70 nm, from about 30 to about 70 nm,from about 40 to about 70 nm, from about 50 to about 70 nm, from about60 to about 70 nm, from about 20 to about 80 nm, from about 30 to about80 nm, from about 40 to about 80 nm, from about 50 to about 80 nm, fromabout 60 to about 80 nm, from about 20 to about 90 nm, from about 30 toabout 90 nm, from about 40 to about 90 nm, from about 50 to about 90 nm,from about 60 to about 90 nm and/or from about 70 to about 90 nm.

In some embodiments, such LNPs are synthesized using methods comprisingmicrofluidic mixers. Exemplary microfluidic mixers can include, but arenot limited to a slit interdigitial micromixer including, but notlimited to those manufactured by Microinnova (Allerheiligen bei Wildon,Austria) and/or a staggered herringbone micromixer (SHM) (Zhigaltsev, I.V. et al., Bottom-up design and synthesis of limit size lipidnanoparticle systems with aqueous and triglyceride cores usingmillisecond microfluidic mixing have been published (Langmuir. 2012.28:3633-40; Belliveau, N. M. et al., Microfluidic synthesis of highlypotent limit-size lipid nanoparticles for in vivo delivery of siRNA.Molecular Therapy-Nucleic Acids. 2012. 1:e37; Chen, D. et al., Rapiddiscovery of potent siRNA-containing lipid nanoparticles enabled bycontrolled microfluidic formulation. J Am Chem Soc. 2012.134(16):6948-51; each of which is herein incorporated by reference inits entirety). In some embodiments, methods of LNP generation comprisingSHM, further comprise the mixing of at least two input streams whereinmixing occurs by microstructure-induced chaotic advection (MICA).According to this method, fluid streams flow through channels present ina herringbone pattern causing rotational flow and folding the fluidsaround each other. This method can also comprise a surface for fluidmixing wherein the surface changes orientations during fluid cycling.Methods of generating LNPs using SHM include those disclosed in U.S.Application Publication Nos. 2004/0262223 and 2012/0276209, each ofwhich is expressly incorporated herein by reference in their entirety.

In one embodiment, the polynucleotide of the present disclosure can beformulated in lipid nanoparticles created using a micromixer such as,but not limited to, a Slit Interdigital Microstructured Mixer (SIMM-V2)or a Standard Slit Interdigital Micro Mixer (SSIMM) or Caterpillar(CPMM) or Impinging-jet (IJMM) from the Institut for Mikrotechnik MainzGmbH, Mainz Germany).

In one embodiment, the polynucleotides of the present disclosure can beformulated in lipid nanoparticles created using microfluidic technology(see Whitesides, George M. The Origins and the Future of Microfluidics.Nature, 2006 442: 368-373; and Abraham et al. Chaotic Mixer forMicrochannels. Science, 2002 295: 647-651; each of which is hereinincorporated by reference in its entirety). As a non-limiting example,controlled microfluidic formulation includes a passive method for mixingstreams of steady pressure-driven flows in micro channels at a lowReynolds number (see, e.g., Abraham et al. Chaotic Mixer forMicrochannels. Science, 2002 295: 647-651; which is herein incorporatedby reference in its entirety).

In one embodiment, the polynucleotides of the present disclosure can beformulated in lipid nanoparticles created using a micromixer chip suchas, but not limited to, those from Harvard Apparatus (Holliston, Mass.)or Dolomite Microfluidics (Royston, UK). A micromixer chip can be usedfor rapid mixing of two or more fluid streams with a split and recombinemechanism.

In one embodiment, the polynucleotides of the disclosure can beformulated for delivery using the drug encapsulating microspheresdescribed in International Patent Publication No. WO2013063468 or U.S.Pat. No. 8,440,614, each of which is herein incorporated by reference inits entirety. The microspheres can comprise a compound of the formula(I), (II), (III), (IV), (V) or (VI) as described in International PatentPublication No. WO2013063468, the contents of which are hereinincorporated by reference in its entirety. In another aspect, the aminoacid, peptide, polypeptide, lipids (APPL) are useful in delivering thepolynucleotides of the disclosure to cells (see International PatentPublication No. WO2013063468, the contents of which is hereinincorporated by reference in its entirety).

In one embodiment, the polynucleotides of the disclosure can beformulated in lipid nanoparticles having a diameter from about 10 toabout 100 nm such as, but not limited to, about 10 to about 20 nm, about10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm,about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 toabout 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm,about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 toabout 60 nm, about 50 to about 70 nm about 50 to about 80 nm, about 50to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm,about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100nm, about 80 to about 90 nm, about 80 to about 100 nm and/or about 90 toabout 100 nm.

In one embodiment, the lipid nanoparticles can have a diameter fromabout 10 to 500 nm.

In one embodiment, the lipid nanoparticle can have a diameter greaterthan 100 nm, greater than 150 nm, greater than 200 nm, greater than 250nm, greater than 300 nm, greater than 350 nm, greater than 400 nm,greater than 450 nm, greater than 500 nm, greater than 550 nm, greaterthan 600 nm, greater than 650 nm, greater than 700 nm, greater than 750nm, greater than 800 nm, greater than 850 nm, greater than 900 nm,greater than 950 nm or greater than 1000 nm.

In one aspect, the lipid nanoparticle can be a limit size lipidnanoparticle described in International Patent Publication No.WO2013059922, the contents of which are herein incorporated by referencein its entirety. The limit size lipid nanoparticle can comprise a lipidbilayer surrounding an aqueous core or a hydrophobic core; where thelipid bilayer can comprise a phospholipid such as, but not limited to,diacylphosphatidylcholine, a diacylphosphatidylethanolamine, a ceramide,a sphingomyelin, a dihydrosphingomyelin, a cephalin, a cerebroside, aC8-C20 fatty acid diacylphophatidylcholine, and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC). In another aspect the limit size lipidnanoparticle can comprise a polyethylene glycol-lipid such as, but notlimited to, DLPE-PEG, DMPE-PEG, DPPC-PEG and DSPE-PEG.

In one embodiment, the polynucleotides can be delivered, localizedand/or concentrated in a specific location using the delivery methodsdescribed in International Patent Publication No. WO2013063530, thecontents of which are herein incorporated by reference in its entirety.As a non-limiting example, a subject can be administered an emptypolymeric particle prior to, simultaneously with or after delivering thepolynucleotides to the subject. The empty polymeric particle undergoes achange in volume once in contact with the subject and becomes lodged,embedded, immobilized or entrapped at a specific location in thesubject.

In one embodiment, the polynucleotides can be formulated in an activesubstance release system (see, e.g., US Patent Publication No.US20130102545, the contents of which is herein incorporated by referencein its entirety). The active substance release system can comprise 1) atleast one nanoparticle bonded to an oligonucleotide inhibitor strandthat is hybridized with a catalytically active nucleic acid and 2) acompound bonded to at least one substrate molecule bonded to atherapeutically active substance (e.g., polynucleotides describedherein), where the therapeutically active substance is released by thecleavage of the substrate molecule by the catalytically active nucleicacid.

In one embodiment, the polynucleotides can be formulated in ananoparticle comprising an inner core comprising a non-cellular materialand an outer surface comprising a cellular membrane. The cellularmembrane can be derived from a cell or a membrane derived from a virus.As a non-limiting example, the nanoparticle can be made by the methodsdescribed in International Patent Publication No. WO2013052167, hereinincorporated by reference in its entirety. As another non-limitingexample, the nanoparticle described in International Patent PublicationNo. WO2013052167, herein incorporated by reference in its entirety, canbe used to deliver the polynucleotides described herein.

In one embodiment, the polynucleotides can be formulated in porousnanoparticle-supported lipid bilayers (protocells). Protocells aredescribed in International Patent Publication No. WO2013056132, thecontents of which are herein incorporated by reference in its entirety.

In one embodiment, the polynucleotides described herein can beformulated in polymeric nanoparticles as described in or made by themethods described in U.S. Pat. Nos. 8,420,123 and 8,518,963 and EuropeanPatent No. EP2073848B1, the contents of each of which are hereinincorporated by reference in their entirety. As a non-limiting example,the polymeric nanoparticle can have a high glass transition temperaturesuch as the nanoparticles described in or nanoparticles made by themethods described in U.S. Pat. No. 8,518,963, the contents of which areherein incorporated by reference in its entirety. As anothernon-limiting example, the polymer nanoparticle for oral and parenteralformulations can be made by the methods described in European Patent No.EP2073848B1, the contents of which are herein incorporated by referencein its entirety.

In another embodiment, the polynucleotides described herein can beformulated in nanoparticles used in imaging. The nanoparticles can beliposome nanoparticles such as those described in US Patent PublicationNo US20130129636, herein incorporated by reference in its entirety. As anon-limiting example, the liposome can comprisegadolinium(III)2-{4,7-bis-carboxymethyl-10-[(N,N-distearylamidomethyl-N′-amido-methyl]-1,4,7,10-tetra-azacyclododec-1-yl}-aceticacid and a neutral, fully saturated phospholipid component (see, e.g.,US Patent Publication No US20130129636, the contents of which is hereinincorporated by reference in its entirety).

In one embodiment, the nanoparticles that can be used in the presentdisclosure are formed by the methods described in U.S. PatentApplication No. US20130130348, the contents of which is hereinincorporated by reference in its entirety.

The nanoparticles of the present disclosure can further includenutrients such as, but not limited to, those which deficiencies can leadto health hazards from anemia to neural tube defects (see e.g., thenanoparticles described in International Patent Publication NoWO2013072929, the contents of which is herein incorporated by referencein its entirety). As a non-limiting example, the nutrient can be iron inthe form of ferrous, ferric salts or elemental iron, iodine, folic acid,vitamins or micronutrients.

In one embodiment, the polynucleotides of the present disclosure can beformulated in a swellable nanoparticle. The swellable nanoparticle canbe, but is not limited to, those described in U.S. Pat. No. 8,440,231,the contents of which is herein incorporated by reference in itsentirety. As a non-limiting embodiment, the swellable nanoparticle canbe used for delivery of the polynucleotides of the present disclosure tothe pulmonary system (see, e.g., U.S. Pat. No. 8,440,231, the contentsof which is herein incorporated by reference in its entirety).

The polynucleotides of the present disclosure can be formulated inpolyanhydride nanoparticles such as, but not limited to, those describedin U.S. Pat. No. 8,449,916, the contents of which is herein incorporatedby reference in its entirety.

The nanoparticles and microparticles of the present disclosure can begeometrically engineered to modulate macrophage and/or the immuneresponse. In one aspect, the geometrically engineered particles can havevaried shapes, sizes and/or surface charges in order to incorporated thepolynucleotides of the present disclosure for targeted delivery such as,but not limited to, pulmonary delivery (see, e.g., InternationalPublication No WO2013082111, the contents of which is hereinincorporated by reference in its entirety). Other physical features thegeometrically engineering particles can have include, but are notlimited to, fenestrations, angled arms, asymmetry and surface roughness,charge that can alter the interactions with cells and tissues. As anon-limiting example, nanoparticles of the present disclosure can bemade by the methods described in International Publication NoWO2013082111, the contents of which is herein incorporated by referencein its entirety.

In one embodiment, the nanoparticles of the present disclosure can bewater soluble nanoparticles such as, but not limited to, those describedin International Publication No. WO2013090601, the contents of which isherein incorporated by reference in its entirety. The nanoparticles canbe inorganic nanoparticles that have a compact and zwitterionic ligandin order to exhibit good water solubility. The nanoparticles can alsohave small hydrodynamic diameters (HD), stability with respect to time,pH, and salinity and a low level of non-specific protein binding.

In one embodiment the nanoparticles of the present disclosure can bedeveloped by the methods described in US Patent Publication No.US20130172406, the contents of which are herein incorporated byreference in its entirety.

In one embodiment, the nanoparticles of the present disclosure arestealth nanoparticles or target-specific stealth nanoparticles such as,but not limited to, those described in US Patent Publication No.US20130172406; the contents of which is herein incorporated by referencein its entirety. The nanoparticles of the present disclosure can be madeby the methods described in US Patent Publication No. US20130172406, thecontents of which are herein incorporated by reference in its entirety.

In another embodiment, the stealth or target-specific stealthnanoparticles can comprise a polymeric matrix. The polymeric matrix cancomprise two or more polymers such as, but not limited to,polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids,polypropylfumerates, polycaprolactones, polyamides, polyacetals,polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinylalcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polycyanoacrylates, polyureas, polystyrenes,polyamines, polyesters, polyanhydrides, polyethers, polyurethanes,polymethacrylates, polyacrylates, polycyanoacrylates or combinationsthereof.

In one embodiment, the nanoparticle can be a nanoparticle-nucleic acidhybrid structure having a high density nucleic acid layer. As anon-limiting example, the nanoparticle-nucleic acid hybrid structure canmade by the methods described in US Patent Publication No.US20130171646, the contents of which are herein incorporated byreference in its entirety. The nanoparticle can comprise a nucleic acidsuch as, but not limited to, polynucleotides described herein and/orknown in the art.

At least one of the nanoparticles of the present disclosure can beembedded in in the core a nanostructure or coated with a low densityporous 3-D structure or coating that is capable of carrying orassociating with at least one payload within or on the surface of thenanostructure. Non-limiting examples of the nanostructures comprising atleast one nanoparticle are described in International Patent PublicationNo. WO2013123523, the contents of which are herein incorporated byreference in its entirety.

Amino Acid Lipids

The disclosure also includes pharmaceutical compositions that comprise aformulation of the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide, with aminoacid lipids. Amino acid lipids are lipophilic compounds comprising anamino acid residue and one or more lipophilic tails. Non-limitingexamples of amino acid lipids and methods of making amino acid lipidsare described in U.S. Pat. No. 8,501,824, the contents of which areherein incorporated by reference in its entirety.

In some embodiments, the amino acid lipids have a hydrophilic portionand a lipophilic portion. The hydrophilic portion can be an amino acidresidue and a lipophilic portion can comprise at least one lipophilictail.

In some embodiments, the amino acid lipid formulations can be used todeliver the polynucleotides to a subject.

In another embodiment, the amino acid lipid formulations can deliver apolynucleotide in releasable form that comprises an amino acid lipidthat binds and releases the polynucleotides. As a non-limiting example,the release of the polynucleotides can be provided by an acid-labilelinker such as, but not limited to, those described in U.S. Pat. Nos.7,098,032, 6,897,196, 6,426,086, 7,138,382, 5,563,250, and 5,505,931,the contents of each of which are herein incorporated by reference inits entirety.

Polymers, Biodegradable Nanoparticles, and Core-Shell Nanoparticles

The disclosure also includes pharmaceutical compositions that comprise aformulation of the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide, usingnatural and/or synthetic polymers. Non-limiting examples of polymersthat can be used for delivery include, but are not limited to, DYNAMICPOLYCONJUGATE® (Arrowhead Research Corp., Pasadena, Calif.) formulationsfrom MIRUS® Bio (Madison, Wis.) and Roche Madison (Madison, Wis.),PHASERX™ polymer formulations such as, without limitation, SMARTTPOLYMER TECHNOLOGY™ (PHASERX®, Seattle, Wash.), DMRI/DOPE, poloxamer,VAXFECTIN® adjuvant from Vical (San Diego, Calif.), chitosan,cyclodextrin from Calando Pharmaceuticals (Pasadena, Calif.), dendrimersand poly(lactic-co-glycolic acid) (PLGA) polymers. RONDEL™(RNAi/Oligonucleotide Nanoparticle Delivery) polymers (ArrowheadResearch Corporation, Pasadena, Calif.) and pH responsive co-blockpolymers such as, but not limited to, PHASERX® (Seattle, Wash.).

A non-limiting example of chitosan formulation includes a core ofpositively charged chitosan and an outer portion of negatively chargedsubstrate (U.S. Pub. No. 20120258176; herein incorporated by referencein its entirety). Chitosan includes, but is not limited to N-trimethylchitosan, mono-N-carboxymethyl chitosan (MCC), N-palmitoyl chitosan(NPCS), EDTA-chitosan, low molecular weight chitosan, chitosanderivatives, or combinations thereof.

In some embodiments, the polymers used in the present disclosure haveundergone processing to reduce and/or inhibit the attachment of unwantedsubstances such as, but not limited to, bacteria, to the surface of thepolymer. The polymer can be processed by methods known and/or describedin the art and/or described in International Pub. No. WO2012150467,herein incorporated by reference in its entirety.

A non-limiting example of PLGA formulations include, but are not limitedto, PLGA injectable depots (e.g., ELIGARD® which is formed by dissolvingPLGA in 66% N-methyl-2-pyrrolidone (NMP) and the remainder being aqueoussolvent and leuprolide. Once injected, the PLGA and leuprolide peptideprecipitates into the subcutaneous space).

Many of these polymer approaches have demonstrated efficacy indelivering oligonucleotides in vivo into the cell cytoplasm (reviewed indeFougerolles Hum Gene Ther. 2008 19:125-132; herein incorporated byreference in its entirety). Two polymer approaches that have yieldedrobust in vivo delivery of nucleic acids, in this case with smallinterfering RNA (siRNA), are dynamic polyconjugates andcyclodextrin-based nanoparticles (see, e.g., US Patent Publication No.US20130156721, herein incorporated by reference in its entirety). Thefirst of these delivery approaches uses dynamic polyconjugates and hasbeen shown in vivo in mice to effectively deliver siRNA and silenceendogenous target mRNA in hepatocytes (Rozema et al., Proc Natl Acad SciUSA. 2007 104:12982-12887; herein incorporated by reference in itsentirety). This particular approach is a multicomponent polymer systemwhose key features include a membrane-active polymer to which nucleicacid, in this case siRNA, is covalently coupled via a disulfide bond andwhere both PEG (for charge masking) and N-acetylgalactosamine (forhepatocyte targeting) groups are linked via pH-sensitive bonds (Rozemaet al., Proc Natl Acad Sci USA. 2007 104:12982-12887; hereinincorporated by reference in its entirety). On binding to the hepatocyteand entry into the endosome, the polymer complex disassembles in thelow-pH environment, with the polymer exposing its positive charge,leading to endosomal escape and cytoplasmic release of the siRNA fromthe polymer. Through replacement of the N-acetylgalactosamine group witha mannose group, it was shown one could alter targeting fromasialoglycoprotein receptor-expressing hepatocytes to sinusoidalendothelium and Kupffer cells. Another polymer approach involves usingtransferrin-targeted cyclodextrin-containing polycation nanoparticles.These nanoparticles have demonstrated targeted silencing of the EWS-FLI1gene product in transferrin receptor-expressing Ewing's sarcoma tumorcells (Hu-Lieskovan et al., Cancer Res. 2005 65: 8984-8982; hereinincorporated by reference in its entirety) and siRNA formulated in thesenanoparticles was well tolerated in non-human primates (Heidel et al.,Proc Natl Acad Sci USA 2007 104:5715-21; herein incorporated byreference in its entirety). Both of these delivery strategiesincorporate rational approaches using both targeted delivery andendosomal escape mechanisms.

The polymer formulation can permit the sustained or delayed release ofpolynucleotides (e.g., following intramuscular or subcutaneousinjection). The altered release profile for the polynucleotide canresult in, for example, translation of an encoded protein over anextended period of time. The polymer formulation can also be used toincrease the stability of the polynucleotide. Biodegradable polymershave been previously used to protect nucleic acids other thanpolynucleotide from degradation and been shown to result in sustainedrelease of payloads in vivo (Rozema et al., Proc Natl Acad Sci USA. 2007104:12982-12887; Sullivan et al., Expert Opin Drug Deliv. 20107:1433-1446; Convertine et al., Biomacromolecules. 2010 Oct. 1; Chu etal., Acc Chem Res. 2012 Jan. 13; Manganiello et al., Biomaterials. 201233:2301-2309; Benoit et al., Biomacromolecules. 2011 12:2708-2714;Singha et al., Nucleic Acid Ther. 2011 2:133-147; deFougerolles Hum GeneTher. 2008 19:125-132; Schaffert and Wagner, Gene Ther. 200816:1131-1138; Chaturvedi et al., Expert Opin Drug Deliv. 20118:1455-1468; Davis, Mol Pharm. 2009 6:659-668; Davis, Nature 2010464:1067-1070; each of which is herein incorporated by reference in itsentirety).

In some embodiments, the pharmaceutical compositions can be sustainedrelease formulations. In a further embodiment, the sustained releaseformulations can be for subcutaneous delivery. Sustained releaseformulations can include, but are not limited to, PLGA microspheres,ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics,Inc. Alachua, Fla.), HYLENEX® (Halozyme Therapeutics, San Diego Calif.),surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia,Ga.), TISSELL® (Baxter International, Inc. Deerfield, Ill.), PEG-basedsealants, and COSEAL® (Baxter International, Inc. Deerfield, Ill.).

As a non-limiting example modified mRNA can be formulated in PLGAmicrospheres by preparing the PLGA microspheres with tunable releaserates (e.g., days and weeks) and encapsulating the modified mRNA in thePLGA microspheres while maintaining the integrity of the modified mRNAduring the encapsulation process. EVAc are non-biodegradable,biocompatible polymers that are used extensively in pre-clinicalsustained release implant applications (e.g., extended release productsOcusert a pilocarpine ophthalmic insert for glaucoma or progestasert asustained release progesterone intrauterine device; transdermal deliverysystems Testoderm, Duragesic and Selegiline; catheters). Poloxamer F-407NF is a hydrophilic, non-ionic surfactant triblock copolymer ofpolyoxyethylene-polyoxypropylene-polyoxyethylene having a low viscosityat temperatures less than 5° C. and forms a solid gel at temperaturesgreater than 15° C. PEG-based surgical sealants comprise two syntheticPEG components mixed in a delivery device that can be prepared in oneminute, seals in 3 minutes and is reabsorbed within 30 days. GELSITE®and natural polymers are capable of in-situ gelation at the site ofadministration. They have been shown to interact with protein andpeptide therapeutic candidates through ionic interaction to provide astabilizing effect.

Polymer formulations can also be selectively targeted through expressionof different ligands as exemplified by, but not limited by, folate,transferrin, and N-acetylgalactosamine (GalNAc) (Benoit et al.,Biomacromolecules. 2011 12:2708-2714; Rozema et al., Proc Natl Acad SciUSA. 2007 104:12982-12887; Davis, Mol Pharm. 2009 6:659-668; Davis,Nature 2010 464:1067-1070; each of which is herein incorporated byreference in its entirety).

The polynucleotides of the disclosure can be formulated with or in apolymeric compound. The polymer can include at least one polymer suchas, but not limited to, polyethenes, polyethylene glycol (PEG),poly(1-lysine)(PLL), PEG grafted to PLL, cationic lipopolymer,biodegradable cationic lipopolymer, polyethyleneimine (PEI),cross-linked branched poly(alkylene imines), a polyamine derivative, amodified poloxamer, a biodegradable polymer, elastic biodegradablepolymer, biodegradable block copolymer, biodegradable random copolymer,biodegradable polyester copolymer, biodegradable polyester blockcopolymer, biodegradable polyester block random copolymer, multiblockcopolymers, linear biodegradable copolymer,poly[α-(4-aminobutyl)-L-glycolic acid) (PAGA), biodegradablecross-linked cationic multi-block copolymers, polycarbonates,polyanhydrides, polyhydroxyacids, polypropylfumerates,polycaprolactones, polyamides, polyacetals, polyethers, polyesters,poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine,poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester), acrylic polymers, amine-containingpolymers, dextran polymers, dextran polymer derivatives or combinationsthereof.

As a non-limiting example, the polynucleotides of the disclosure can beformulated with the polymeric compound of PEG grafted with PLL asdescribed in U.S. Pat. No. 6,177,274; herein incorporated by referencein its entirety. The formulation can be used for transfecting cells invitro or for in vivo delivery of polynucleotide. In another example, thepolynucleotide can be suspended in a solution or medium with a cationicpolymer, in a dry pharmaceutical composition or in a solution that iscapable of being dried as described in U.S. Pub. Nos. 20090042829 and20090042825; each of which are herein incorporated by reference in theirentireties.

As another non-limiting example the polynucleotides of the disclosurecan be formulated with a PLGA-PEG block copolymer (see US Pub. No.US20120004293 and U.S. Pat. No. 8,236,330, herein incorporated byreference in their entireties) or PLGA-PEG-PLGA block copolymers (SeeU.S. Pat. No. 6,004,573, herein incorporated by reference in itsentirety). As a non-limiting example, the polynucleotides of thedisclosure can be formulated with a diblock copolymer of PEG and PLA orPEG and PLGA (see U.S. Pat. No. 8,246,968, herein incorporated byreference in its entirety).

A polyamine derivative can be used to deliver nucleic acids or to treatand/or prevent a disease or to be included in an implantable orinjectable device (U.S. Pub. No. 20100260817 (now U.S. Pat. No.8,460,696) the contents of each of which is herein incorporated byreference in its entirety). As a non-limiting example, a pharmaceuticalcomposition can include the polynucleotide and the polyamine derivativedescribed in U.S. Pub. No. 20100260817 (now U.S. Pat. No. 8,460,696; thecontents of which are incorporated herein by reference in its entirety.As a non-limiting example the polynucleotides of the present disclosurecan be delivered using a polyamine polymer such as, but not limited to,a polymer comprising a 1,3-dipolar addition polymer prepared bycombining a carbohydrate diazide monomer with a dialkyne unitecomprising oligoamines (U.S. Pat. No. 8,236,280; herein incorporated byreference in its entirety).

The polynucleotides of the disclosure can be formulated with at leastone acrylic polymer. Acrylic polymers include but are not limited to,acrylic acid, methacrylic acid, acrylic acid and methacrylic acidcopolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates,cyanoethyl methacrylate, amino alkyl methacrylate copolymer,poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates andcombinations thereof.

In some embodiments, the polynucleotides of the present disclosure canbe formulated with at least one polymer and/or derivatives thereofdescribed in International Publication Nos. WO2011115862, WO2012082574and WO2012068187 and U.S. Pub. No. 20120283427, each of which are hereinincorporated by reference in their entireties. In another embodiment,the polynucleotides of the present disclosure can be formulated with apolymer of formula Z as described in WO2011115862, herein incorporatedby reference in its entirety. In yet another embodiment, thepolynucleotides can be formulated with a polymer of formula Z, Z′ or Z″as described in International Pub. Nos. WO2012082574 or WO2012068187 andU.S. Pub. No. 2012028342, each of which are herein incorporated byreference in their entireties. The polymers formulated with the modifiedRNA of the present disclosure can be synthesized by the methodsdescribed in International Pub. Nos. WO2012082574 or WO2012068187, eachof which are herein incorporated by reference in their entireties.

The polynucleotides of the disclosure can be formulated with at leastone acrylic polymer. Acrylic polymers include but are not limited to,acrylic acid, methacrylic acid, acrylic acid and methacrylic acidcopolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates,cyanoethyl methacrylate, amino alkyl methacrylate copolymer,poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates andcombinations thereof.

Formulations of polynucleotides of the disclosure can include at leastone amine-containing polymer such as, but not limited to polylysine,polyethylene imine, poly(amidoamine) dendrimers, poly(amine-co-esters)or combinations thereof. As a non-limiting example, thepoly(amine-co-esters) can be the polymers described in and/or made bythe methods described in International Publication No WO2013082529, thecontents of which are herein incorporated by reference in its entirety.

For example, the polynucleotides of the disclosure can be formulated ina pharmaceutical compound including a poly(alkylene imine), abiodegradable cationic lipopolymer, a biodegradable block copolymer, abiodegradable polymer, or a biodegradable random copolymer, abiodegradable polyester block copolymer, a biodegradable polyesterpolymer, a biodegradable polyester random copolymer, a linearbiodegradable copolymer, PAGA, a biodegradable cross-linked cationicmulti-block copolymer or combinations thereof. The biodegradablecationic lipopolymer can be made by methods known in the art and/ordescribed in U.S. Pat. No. 6,696,038, U.S. App. Nos. 20030073619 and20040142474 each of which is herein incorporated by reference in theirentireties. The poly(alkylene imine) can be made using methods known inthe art and/or as described in U.S. Pub. No. 20100004315, hereinincorporated by reference in its entirety. The biodegradable polymer,biodegradable block copolymer, the biodegradable random copolymer,biodegradable polyester block copolymer, biodegradable polyesterpolymer, or biodegradable polyester random copolymer can be made usingmethods known in the art and/or as described in U.S. Pat. Nos. 6,517,869and 6,267,987, the contents of which are each incorporated herein byreference in their entirety. The linear biodegradable copolymer can bemade using methods known in the art and/or as described in U.S. Pat. No.6,652,886. The PAGA polymer can be made using methods known in the artand/or as described in U.S. Pat. No. 6,217,912 herein incorporated byreference in its entirety. The PAGA polymer can be copolymerized to forma copolymer or block copolymer with polymers such as but not limited to,poly-L-lysine, polyarginine, polyornithine, histones, avidin,protamines, polylactides and poly(lactide-co-glycolides). Thebiodegradable cross-linked cationic multi-block copolymers can be mademy methods known in the art and/or as described in U.S. Pat. Nos.8,057,821, 8,444,992 or U.S. Pub. No. 2012009145 each of which areherein incorporated by reference in their entireties. For example, themulti-block copolymers can be synthesized using linear polyethyleneimine(LPEI) blocks that have distinct patterns as compared to branchedpolyethyleneimines. Further, the composition or pharmaceuticalcomposition can be made by the methods known in the art, describedherein, or as described in U.S. Pub. No. 20100004315 or U.S. Pat. Nos.6,267,987 and 6,217,912 each of which are herein incorporated byreference in their entireties.

The polynucleotides of the disclosure can be formulated with at leastone degradable polyester that can contain polycationic side chains.Degradable polyesters include, but are not limited to, poly(serineester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester),and combinations thereof. In another embodiment, the degradablepolyesters can include a PEG conjugation to form a PEGylated polymer.

The polynucleotides of the disclosure can be formulated with at leastone crosslinkable polyester. Crosslinkable polyesters include thoseknown in the art and described in US Pub. No. 20120269761, the contentsof which is herein incorporated by reference in its entirety.

The polynucleotides of the disclosure can be formulated in or with atleast one cyclodextrin polymer. Cyclodextrin polymers and methods ofmaking cyclodextrin polymers include those known in the art anddescribed in US Pub. No. 20130184453, the contents of which are hereinincorporated by reference in its entirety.

In some embodiments, the polynucleotides of the disclosure can beformulated in or with at least one crosslinked cation-binding polymers.Crosslinked cation-binding polymers and methods of making crosslinkedcation-binding polymers include those known in the art and described inInternational Patent Publication No. WO2013106072, WO2013106073 andWO2013106086, the contents of each of which are herein incorporated byreference in its entirety.

In some embodiments, the polynucleotides of the disclosure can beformulated in or with at least one branched polymer. Branched polymersand methods of making branched polymers include those known in the artand described in International Patent Publication No. WO2013113071, thecontents of each of which are herein incorporated by reference in itsentirety.

In some embodiments, the polynucleotides of the disclosure can beformulated in or with at least PEGylated albumin polymer. PEGylatedalbumin polymer and methods of making PEGylated albumin polymer includethose known in the art and described in US Patent Publication No.US20130231287, the contents of each of which are herein incorporated byreference in its entirety.

In some embodiments, the polymers described herein can be conjugated toa lipid-terminating PEG. As a non-limiting example, PLGA can beconjugated to a lipid-terminating PEG forming PLGA-DSPE-PEG. As anothernon-limiting example, PEG conjugates for use with the present disclosureare described in International Publication No. WO2008103276, hereinincorporated by reference in its entirety. The polymers can beconjugated using a ligand conjugate such as, but not limited to, theconjugates described in U.S. Pat. No. 8,273,363, herein incorporated byreference in its entirety.

In some embodiments, the polynucleotides disclosed herein can be mixedwith the PEGs or the sodium phosphate/sodium carbonate solution prior toadministration. In another embodiment, a polynucleotides encoding aprotein of interest can be mixed with the PEGs and also mixed with thesodium phosphate/sodium carbonate solution. In yet another embodiment,polynucleotides encoding a protein of interest can be mixed with thePEGs and a polynucleotides encoding a second protein of interest can bemixed with the sodium phosphate/sodium carbonate solution.

In some embodiments, the polynucleotides described herein can beconjugated with another compound. Non-limiting examples of conjugatesare described in U.S. Pat. Nos. 7,964,578 and 7,833,992, each of whichare herein incorporated by reference in their entireties. In anotherembodiment, modified RNA of the present disclosure can be conjugatedwith conjugates of formula 1-122 as described in U.S. Pat. Nos.7,964,578 and 7,833,992, each of which are herein incorporated byreference in their entireties. The polynucleotides described herein canbe conjugated with a metal such as, but not limited to, gold. (See,e.g., Giljohann et al. Journ. Amer. Chem. Soc. 2009 131(6): 2072-2073;herein incorporated by reference in its entirety). In anotherembodiment, the polynucleotides described herein can be conjugatedand/or encapsulated in gold-nanoparticles. (International Pub. No.WO201216269 and U.S. Pub. No. 20120302940 and US20130177523; thecontents of each of which is herein incorporated by reference in itsentirety).

As described in U.S. Pub. No. 20100004313, herein incorporated byreference in its entirety, a gene delivery composition can include anucleotide sequence and a poloxamer. For example, the polynucleotides ofthe present disclosure can be used in a gene delivery composition withthe poloxamer described in U.S. Pub. No. 20100004313.

In some embodiments, the polymer formulation of the present disclosurecan be stabilized by contacting the polymer formulation, which caninclude a cationic carrier, with a cationic lipopolymer that can becovalently linked to cholesterol and polyethylene glycol groups. Thepolymer formulation can be contacted with a cationic lipopolymer usingthe methods described in U.S. Pub. No. 20090042829 herein incorporatedby reference in its entirety. The cationic carrier can include, but isnot limited to, polyethylenimine, poly(trimethylenimine),poly(tetramethylenimine), polypropylenimine, aminoglycoside-polyamine,dideoxy-diamino-b-cyclodextrin, spermine, spermidine,poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine),poly(arginine), cationized gelatin, dendrimers, chitosan,1,2-Dioleoyl-3-Trimethylammonium-Propane(DOTAP),N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride(DOTIM),2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA),3B—[N—(N′,N′-Dimethylaminoethane)-carbamoyl]Cholesterol Hydrochloride(DC-Cholesterol HCl) diheptadecylamidoglycyl spermidine (DOGS), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride DODAC) andcombinations thereof. As a non-limiting example, the polynucleotides canbe formulated with a cationic lipopolymer such as those described inU.S. Patent Application No. 20130065942, herein incorporated byreference in its entirety.

The polynucleotides of the disclosure can be formulated in a polyplex ofone or more polymers (See, e.g., U.S. Pat. No. 8,501,478, U.S. Pub. No.20120237565 and 20120270927 and 20130149783 and International PatentPub. No. WO2013090861; the contents of each of which is hereinincorporated by reference in its entirety). As a non-limiting example,the polyplex can be formed using the novel alpha-aminoamidine polymersdescribed in International Publication No. WO2013090861, the contents ofwhich are herein incorporated by reference in its entirety. As anothernon-limiting example, the polyplex can be formed using the clickpolymers described in U.S. Pat. No. 8,501,478, the contents of which isherein incorporated by reference in its entirety.

In some embodiments, the polyplex comprises two or more cationicpolymers. The cationic polymer can comprise a poly(ethylene imine) (PEI)such as linear PEI. In another embodiment, the polyplex comprisesp(TETA/CBA) its PEGylated analog p(TETA/CBA)-g-PEG2k and mixturesthereof (see, e.g., US Patent Publication No. US20130149783, thecontents of which are herein incorporated by reference in its entirety.

The polynucleotides of the disclosure can also be formulated as ananoparticle using a combination of polymers, lipids, and/or otherbiodegradable agents, such as, but not limited to, calcium phosphate.Components can be combined in a core-shell, hybrid, and/orlayer-by-layer architecture, to allow for fine-tuning of thenanoparticle so to delivery of the polynucleotide, polynucleotides canbe enhanced (Wang et al., Nat Mater. 2006 5:791-796; Fuller et al.,Biomaterials. 2008 29:1526-1532; DeKoker et al., Adv Drug Deliv Rev.2011 63:748-761; Endres et al., Biomaterials. 2011 32:7721-7731; Su etal., Mol Pharm. 2011 Jun. 6; 8(3):774-87; herein incorporated byreference in its entirety). As a non-limiting example, the nanoparticlecan comprise a plurality of polymers such as, but not limited tohydrophilic-hydrophobic polymers (e.g., PEG-PLGA), hydrophobic polymers(e.g., PEG) and/or hydrophilic polymers (International Pub. No.WO20120225129; the contents of which is herein incorporated by referencein its entirety).

As another non-limiting example the nanoparticle comprising hydrophilicpolymers for the polynucleotides can be those described in or made bythe methods described in International Patent Publication No.WO2013119936, the contents of which are herein incorporated by referencein its entirety.

In some embodiments, the biodegradable polymers that can be used in thepresent disclosure are poly(ether-anhydride) block copolymers. As anon-limiting example, the biodegradable polymers used herein can be ablock copolymer as described in International Patent Publication NoWO2006063249, herein incorporated by reference in its entirety, or madeby the methods described in International Patent Publication NoWO2006063249, herein incorporated by reference in its entirety.

In another embodiment, the biodegradable polymers that can be used inthe present disclosure are alkyl and cycloalkyl terminated biodegradablelipids. As a non-limiting example, the alkyl and cycloalkyl terminatedbiodegradable lipids can be those described in International PublicationNo. WO2013086322 and/or made by the methods described in InternationalPublication No. WO2013086322; the contents of which are hereinincorporated by reference in its entirety.

In yet another embodiment, the biodegradable polymers that can be usedin the present disclosure are cationic lipids having one or morebiodegradable group located in a lipid moiety. As a non-limitingexample, the biodegradable lipids can be those described in US PatentPublication No. US20130195920, the contents of which are hereinincorporated by reference in its entirety.

Biodegradable calcium phosphate nanoparticles in combination with lipidsand/or polymers have been shown to deliver polynucleotides in vivo. Insome embodiments, a lipid coated calcium phosphate nanoparticle, whichcan also contain a targeting ligand such as anisamide, can be used todeliver the polynucleotide, polynucleotides of the present disclosure.For example, to effectively deliver siRNA in a mouse metastatic lungmodel a lipid coated calcium phosphate nanoparticle was used (Li et al.,J Contr Rel. 2010 142: 416-421; Li et al., J Contr Rel. 2012158:108-114; Yang et al., Mol Ther. 2012 20:609-615; herein incorporatedby reference in its entirety). This delivery system combines both atargeted nanoparticle and a component to enhance the endosomal escape,calcium phosphate, in order to improve delivery of the siRNA.

In some embodiments, calcium phosphate with a PEG-polyanion blockcopolymer can be used to delivery polynucleotides (Kazikawa et al., JContr Rel. 2004 97:345-356; Kazikawa et al., J Contr Rel. 2006111:368-370; the contents of each of which are herein incorporated byreference in its entirety).

In some embodiments, a PEG-charge-conversional polymer (Pitella et al.,Biomaterials. 2011 32:3106-3114; the contents of which are hereinincorporated by reference in its entirety) can be used to form ananoparticle to deliver the polynucleotides of the present disclosure.The PEG-charge-conversional polymer can improve upon the PEG-polyanionblock copolymers by being cleaved into a polycation at acidic pH, thusenhancing endosomal escape.

In some embodiments, a polymer used in the present disclosure can be apentablock polymer such as, but not limited to, the pentablock polymersdescribed in International Patent Publication No. WO2013055331, hereinincorporated by reference in its entirety. As a non-limiting example,the pentablock polymer comprises PGA-PCL-PEG-PCL-PGA, wherein PEG ispolyethylene glycol, PCL is poly(E-caprolactone), PGA is poly(glycolicacid), and PLA is poly(lactic acid). As another non-limiting example,the pentablock polymer comprises PEG-PCL-PLA-PCL-PEG, wherein PEG ispolyethylene glycol, PCL is poly(E-caprolactone), PGA is poly(glycolicacid), and PLA is poly(lactic acid).

In some embodiments, a polymer that can be used in the presentdisclosure comprises at least one diepoxide and at least oneaminoglycoside (See, e.g., International Patent Publication No.WO2013055971, the contents of which are herein incorporated by referencein its entirety). The diepoxide can be selected from, but is not limitedto, 1,4 butanediol diglycidyl ether (1,4 B), 1,4-cyclohexanedimethanoldiglycidyl ether (1,4 C), 4-vinylcyclohexene diepoxide (4VCD),ethyleneglycol diglycidyl ether (EDGE), glycerol diglycidyl ether (GDE),neopentylglycol diglycidyl ether (NPDGE), poly(ethyleneglycol)diglycidyl ether (PEGDE), poly(propyleneglycol) diglycidyl ether (PPGDE)and resorcinol diglycidyl ether (RDE). The aminoglycoside can beselected from, but is not limited to, streptomycin, neomycin,framycetin, paromomycin, ribostamycin, kanamycin, amikacin, arbekacin,bekanamycin, dibekacin, tobramycin, spectinomycin, hygromycin,gentamicin, netilmicin, sisomicin, isepamicin, verdamicin, astromicin,and apramycin. As a non-limiting example, the polymers can be made bythe methods described in International Patent Publication No.WO2013055971, the contents of which are herein incorporated by referencein its entirety. As another non-limiting example, compositionscomprising any of the polymers comprising at least one least onediepoxide and at least one aminoglycoside can be made by the methodsdescribed in International Patent Publication No. WO2013055971, thecontents of which are herein incorporated by reference in its entirety.

In some embodiments, a polymer that can be used in the presentdisclosure can be a cross-linked polymer. As a non-limiting example, thecross-linked polymers can be used to form a particle as described inU.S. Pat. No. 8,414,927, the contents of which are herein incorporatedby reference in its entirety. As another non-limiting example, thecross-linked polymer can be obtained by the methods described in USPatent Publication No. US20130172600, the contents of which are hereinincorporated by reference in its entirety.

In another embodiment, a polymer that can be used in the presentdisclosure can be a cross-linked polymer such as those described in U.S.Pat. No. 8,461,132, the contents of which are herein incorporated byreference in its entirety. As a non-limiting example, the cross-linkedpolymer can be used in a therapeutic composition for the treatment of abody tissue. The therapeutic composition can be administered to damagedtissue using various methods known in the art and/or described hereinsuch as injection or catheterization.

In some embodiments, a polymer that can be used in the presentdisclosure can be a di-alphatic substituted pegylated lipid such as, butnot limited to, those described in International Patent Publication No.WO2013049328, the contents of which are herein incorporated by referencein its entirety.

In some embodiments, a block copolymer is PEG-PLGA-PEG (see, e.g., thethermosensitive hydrogel (PEG-PLGA-PEG) was used as a TGF-beta1 genedelivery vehicle in Lee et al. Thermosensitive Hydrogel as a Tgf-β1 GeneDelivery Vehicle Enhances Diabetic Wound Healing. PharmaceuticalResearch, 2003 20(12): 1995-2000; as a controlled gene delivery systemin Li et al. Controlled Gene Delivery System Based on ThermosensitiveBiodegradable Hydrogel. Pharmaceutical Research 2003 20(6):884-888; andChang et al., Non-ionic amphiphilic biodegradable PEG-PLGA-PEG copolymerenhances gene delivery efficiency in rat skeletal muscle. J ControlledRelease. 2007 118:245-253; each of which is herein incorporated byreference in its entirety) can be used in the present disclosure. Thepresent disclosure can be formulated with PEG-PLGA-PEG foradministration such as, but not limited to, intramuscular andsubcutaneous administration.

In another embodiment, the PEG-PLGA-PEG block copolymer is used in thepresent disclosure to develop a biodegradable sustained release system.In one aspect, the polynucleotides of the present disclosure are mixedwith the block copolymer prior to administration. In another aspect, thepolynucleotides acids of the present disclosure are co-administered withthe block copolymer.

In some embodiments, the polymer used in the present disclosure can be amulti-functional polymer derivative such as, but not limited to, amulti-functional N-maleimidyl polymer derivatives as described in U.S.Pat. No. 8,454,946, the contents of which are herein incorporated byreference in its entirety.

The use of core-shell nanoparticles has additionally focused on ahigh-throughput approach to synthesize cationic cross-linked nanogelcores and various shells (Siegwart et al., Proc Natl Acad Sci USA. 2011108:12996-13001; the contents of which are herein incorporated byreference in its entirety). The complexation, delivery, andinternalization of the polymeric nanoparticles can be preciselycontrolled by altering the chemical composition in both the core andshell components of the nanoparticle. For example, the core-shellnanoparticles can efficiently deliver siRNA to mouse hepatocytes afterthey covalently attach cholesterol to the nanoparticle.

In some embodiments, a hollow lipid core comprising a middle PLGA layerand an outer neutral lipid layer containing PEG can be used to deliveryof the polynucleotide, polynucleotides of the present disclosure. As anon-limiting example, in mice bearing a luciferease-expressing tumor, itwas determined that the lipid-polymer-lipid hybrid nanoparticlesignificantly suppressed luciferase expression, as compared to aconventional lipoplex (Shi et al, Angew Chem Int Ed. 2011 50:7027-7031;herein incorporated by reference in its entirety).

In some embodiments, the lipid nanoparticles can comprise a core of thepolynucleotides disclosed herein and a polymer shell. The polymer shellcan be any of the polymers described herein and are known in the art. Inan additional embodiment, the polymer shell can be used to protect thepolynucleotides in the core.

Core-shell nanoparticles for use with the polynucleotides of the presentdisclosure are described and can be formed by the methods described inU.S. Pat. No. 8,313,777 or International Patent Publication No.WO2013124867, the contents of each of which are herein incorporated byreference in their entirety.

In some embodiments, the core-shell nanoparticles can comprise a core ofthe polynucleotides disclosed herein and a polymer shell. The polymershell can be any of the polymers described herein and are known in theart. In an additional embodiment, the polymer shell can be used toprotect the polynucleotides in the core.

In some embodiments, the polymer used with the formulations describedherein can be a modified polymer (such as, but not limited to, amodified polyacetal) as described in International Publication No.WO2011120053, the contents of which are herein incorporated by referencein its entirety.

In some embodiments, the formulation can be a polymeric carrier cargocomplex comprising a polymeric carrier and at least one nucleic acidmolecule. Non-limiting examples of polymeric carrier cargo complexes aredescribed in International Patent Publications Nos. WO2013113326,WO2013113501, WO2013113325, WO2013113502 and WO2013113736 and EuropeanPatent Publication No. EP2623121, the contents of each of which areherein incorporated by reference in their entireties. In one aspect thepolymeric carrier cargo complexes can comprise a negatively chargednucleic acid molecule such as, but not limited to, those described inInternational Patent Publication Nos. WO2013113325 and WO2013113502, thecontents of each of which are herein incorporated by reference in itsentirety.

In some embodiments, a pharmaceutical composition can comprisepolynucleotides of the disclosure and a polymeric carrier cargo complex(See, e.g., the antigens described in International Patent PublicationsNos. WO2013113326, WO2013113501, WO2013113325, WO2013113502 andWO2013113736 and European Patent Publication No. EP2623121, the contentsof each of which are herein incorporated by reference in theirentireties).

In some embodiments, the polymer used with the formulations describedherein can be a modified polymer (such as, but not limited to, amodified polyacetal) as described in International Publication No.WO2011120053, the contents of which are herein incorporated by referencein its entirety

Peptides and Proteins

The disclosure also includes pharmaceutical compositions that comprise aformulation of the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide, using oneor more peptides and/or proteins. The polynucleotides of the disclosurecan be formulated with peptides and/or proteins in order to increasetransfection of cells by the polynucleotide. In some embodiments,peptides such as, but not limited to, proteins and peptides that enableintracellular or mitochondrial delivery can be used to deliverpharmaceutical formulations.

Formulations of the disclosure including peptides or proteins can beused to increase cell transfection by the polynucleotide, alter thebiodistribution of the polynucleotide (e.g., by targeting specifictissues or cell types), and/or increase the translation of encodedprotein. (See e.g., International Pub. No. WO2012110636 andWO2013123298; the contents of which are herein incorporated by referencein its entirety).

In some embodiments, the cell penetrating peptide can be, but is notlimited to, those described in US Patent Publication No US20130129726,US20130137644 and US20130164219, each of which is herein incorporated byreference in its entirety.

Self-Assembled Macromolecules

The disclosure also includes pharmaceutical compositions that comprise aformulation of the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide, withamphiphilic macromolecules (AMs) for delivery. AMs comprisebiocompatible amphiphilic polymers that have an alkylated sugar backbonecovalently linked to poly(ethylene glycol). In aqueous solution, the AMsself-assemble to form micelles. Non-limiting examples of methods offorming AMs and AMs are described in US Patent Publication No.US20130217753, the contents of which are herein incorporated byreference in its entirety.

Cations and Anions

The disclosure also includes pharmaceutical compositions that comprise aformulation of the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide, withcations or anions. In some embodiments, the formulations include metalcations such as, but not limited to, Zn2+, Ca2+, Cu2+, Mg+ andcombinations thereof. As a non-limiting example, formulations caninclude polymers and a polynucleotides complexed with a metal cation(see, e.g., U.S. Pat. Nos. 6,265,389 and 6,555,525, each of which isherein incorporated by reference in its entirety).

In some embodiments, cationic nanoparticles comprising combinations ofdivalent and monovalent cations can be formulated with polynucleotides.Such nanoparticles can form spontaneously in solution over a givenperiod (e.g., hours, days, etc.). Such nanoparticles do not form in thepresence of divalent cations alone or in the presence of monovalentcations alone. The delivery of polynucleotides in cationic nanoparticlesor in one or more depot comprising cationic nanoparticles can improvepolynucleotide bioavailability by acting as a long-acting depot and/orreducing the rate of degradation by nucleases.

Suspension Formulations

The disclosure also includes pharmaceutical compositions that comprise aformulation of the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide, insuspensions. In some embodiments, suspension formulations are providedcomprising polynucleotides, water immiscible oil depots, surfactantsand/or cosurfactants and/or co-solvents. Combinations of oils andsurfactants can enable suspension formulation with polynucleotides.Delivery of polynucleotides in a water immiscible depot can be used toimprove bioavailability through sustained release of mRNA from the depotto the surrounding physiologic environment and prevent polynucleotidesdegradation by nucleases.

In some embodiments, suspension formulations of mRNA can be preparedusing combinations of polynucleotides, oil-based solutions andsurfactants. Such formulations can be prepared as a two-part systemcomprising an aqueous phase comprising polynucleotides and an oil-basedphase comprising oil and surfactants. Exemplary oils for suspensionformulations can include, but are not limited to sesame oil and Miglyol(comprising esters of saturated coconut and palmkernel oil-derivedcaprylic and capric fatty acids and glycerin or propylene glycol), cornoil, soybean oil, peanut oil, beeswax and/or palm seed oil. Exemplarysurfactants can include, but are not limited to Cremophor, polysorbate20, polysorbate 80, polyethylene glycol, transcutol, Capmul®, labrasol,isopropyl myristate, and/or Span 80. In some embodiments, suspensionscan comprise co-solvents including, but not limited to ethanol, glyceroland/or propylene glycol.

Suspensions can be formed by first preparing polynucleotides formulationcomprising an aqueous solution of polynucleotide and an oil-based phasecomprising one or more surfactants. Suspension formation occurs as aresult of mixing the two phases (aqueous and oil-based). In someembodiments, such a suspension can be delivered to an aqueous phase toform an oil-in-water emulsion. In some embodiments, delivery of asuspension to an aqueous phase results in the formation of anoil-in-water emulsion in which the oil-based phase comprisingpolynucleotides forms droplets that can range in size fromnanometer-sized droplets to micrometer-sized droplets. In someembodiments, specific combinations of oils, surfactants, cosurfactantsand/or co-solvents can be utilized to suspend polynucleotides in the oilphase and/or to form oil-in-water emulsions upon delivery into anaqueous environment.

In some embodiments, suspensions can provide modulation of the releaseof polynucleotides into the surrounding environment. In suchembodiments, polynucleotides release can be modulated by diffusion froma water immiscible depot followed by resolubilization into a surroundingenvironment (e.g., an aqueous environment).

In some embodiments, polynucleotides within a water immiscible depot(e.g., suspended within an oil phase) can result in alteredpolynucleotides stability (e.g., altered degradation by nucleases).

In some embodiments, polynucleotides can be formulated such that uponinjection, an emulsion forms spontaneously (e.g., when delivered to anaqueous phase). Such particle formation can provide a high surface areato volume ratio for release of polynucleotides from an oil phase to anaqueous phase.

In some embodiments, the polynucleotides can be formulated in ananoemulsion such as, but not limited to, the nanoemulsions described inU.S. Pat. No. 8,496,945, the contents of which are herein incorporatedby reference in its entirety. The nanoemulsions can comprisenanoparticles described herein. As a non-limiting example, thenanoparticles can comprise a liquid hydrophobic core that can besurrounded or coated with a lipid or surfactant layer. The lipid orsurfactant layer can comprise at least one membrane-integrating peptideand can also comprise a targeting ligand (see, e.g., U.S. Pat. No.8,496,945, the contents of which are herein incorporated by reference inits entirety).

Semi-Solid Compositions

The disclosure also includes pharmaceutical compositions that comprise aformulation of the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide, insemi-solid compositions. In some embodiments, the polynucleotides can beformulated with a hydrophobic matrix to form a semi-solid composition.As a non-limiting example, the semi-solid composition or paste-likecomposition can be made by the methods described in International PatentPublication No WO201307604, herein incorporated by reference in itsentirety. The semi-solid composition can be a sustained releaseformulation as described in International Patent Publication NoWO201307604, herein incorporated by reference in its entirety.

In another embodiment, the semi-solid composition can further have amicro-porous membrane or a biodegradable polymer formed around thecomposition (see, e.g., International Patent Publication No WO201307604,herein incorporated by reference in its entirety).

The semi-solid composition using the polynucleotides of the presentdisclosure can have the characteristics of the semi-solid mixture asdescribed in International Patent Publication No WO201307604, hereinincorporated by reference in its entirety (e.g., a modulus of elasticityof at least 10⁻⁴ N·mm⁻², and/or a viscosity of at least 100 mPa·s).

Surgical Sealants: Gels and Hydrogels

The disclosure also includes pharmaceutical compositions that comprise aformulation of the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide, withsurgical sealants. In some embodiments, the polynucleotides disclosedherein can be encapsulated into any hydrogel known in the art that canform a gel when injected into a subject. Surgical sealants such as gelsand hydrogels are described in International Patent Application No.PCT/US2014/027077, the contents of which are herein incorporated byreference in its entirety.

Conjugates

The disclosure includes pharmaceutical compositions that compriseconjugates of the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide. Thepolynucleotides of the disclosure include conjugates, such as apolynucleotide covalently linked to a carrier or targeting group, orincluding two encoding regions that together produce a fusion protein(e.g., bearing a targeting group and therapeutic protein or peptide).

The conjugates of the disclosure include a naturally occurringsubstance, such as a protein (e.g., human serum albumin (HSA),low-density lipoprotein (LDL), high-density lipoprotein (HDL), orglobulin); an carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand canalso be a recombinant or synthetic molecule, such as a syntheticpolymer, e.g., a synthetic polyamino acid, an oligonucleotide (e.g., anaptamer). Examples of polyamino acids include polyamino acid is apolylysine (PLL), poly L-aspartic acid, poly L-glutamic acid,styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied)copolymer, divinyl ether-maleic anhydride copolymer,N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol(PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllicacid), N-isopropylacrylamide polymers, or polyphosphazine. Example ofpolyamines include: polyethylenimine, polylysine (PLL), spermine,spermidine, polyamine, pseudopeptide-polyamine, peptidomimeticpolyamine, dendrimer polyamine, arginine, amidine, protamine, cationiclipid, cationic porphyrin, quaternary salt of a polyamine, or an alphahelical peptide.

In some embodiments, the conjugate of the present disclosure canfunction as a carrier for the polynucleotides of the present disclosure.The conjugate can comprise a cationic polymer such as, but not limitedto, polyamine, polylysine, polyalkylenimine, and polyethylenimine thatcan be grafted to with poly(ethylene glycol). As a non-limiting example,the conjugate can be similar to the polymeric conjugate and the methodof synthesizing the polymeric conjugate described in U.S. Pat. No.6,586,524 herein incorporated by reference in its entirety.

A non-limiting example of a method for conjugation to a substrate isdescribed in US Patent Publication No. US20130211249, the contents ofwhich are herein incorporated by reference in its entirety. The methodcan be used to make a conjugated polymeric particle comprising apolynucleotide.

The conjugates can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGDpeptide mimetic or an aptamer.

Targeting groups can be proteins, e.g., glycoproteins, or peptides,e.g., molecules having a specific affinity for a co-ligand, orantibodies e.g., an antibody, that binds to a specified cell type suchas a cancer cell, endothelial cell, or bone cell. Targeting groups canalso include hormones and hormone receptors. They can also includenon-peptidic species, such as lipids, lectins, carbohydrates, vitamins,cofactors, multivalent lactose, multivalent galactose,N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose,multivalent frucose, or aptamers. The ligand can be, for example, alipopolysaccharide, or an activator of p38 MAP kinase.

The targeting group can be any ligand that is capable of targeting aspecific receptor. Examples include, without limitation, folate, GalNAc,galactose, mannose, mannose-6P, apatamers, integrin receptor ligands,chemokine receptor ligands, transferrin, biotin, serotonin receptorligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. Inparticular embodiments, the targeting group is an aptamer. The aptamercan be unmodified or have any combination of modifications disclosedherein.

As a non-limiting example, the targeting group can be a glutathionereceptor (GR)-binding conjugate for targeted delivery across theblood-central nervous system barrier (See, e.g., US Patent PublicationNo. US2013021661012, the contents of which are herein incorporated byreference in its entirety.

In some embodiments, the conjugate of the present disclosure can be asynergistic biomolecule-polymer conjugate. The synergisticbiomolecule-polymer conjugate can be long-acting continuous-releasesystem to provide a greater therapeutic efficacy. The synergisticbiomolecule-polymer conjugate can be those described in US PatentPublication No. US20130195799, the contents of which are hereinincorporated by reference in its entirety.

In another embodiment, the conjugate that can be used in the presentdisclosure can be an aptamer conjugate. Non-limiting examples ofapatamer conjugates are described in International Patent PublicationNo. WO2012040524, the contents of which are herein incorporated byreference in its entirety. The aptamer conjugates can be used to providetargeted delivery of formulations comprising polynucleotides.

In some embodiments, the conjugate that can be used in the presentdisclosure can be an amine containing polymer conjugate. Non-limitingexamples of amine containing polymer conjugate are described in U.S.Pat. No. 8,507,653, the contents of which are herein incorporated byreference in its entirety.

In some embodiments, pharmaceutical compositions of the presentdisclosure can include chemical modifications such as, but not limitedto, modifications similar to locked nucleic acids.

Representative U.S. patents that teach the preparation of locked nucleicacid (LNA) such as those from Santaris, include, but are not limited to,the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499;6,998,484; 7,053,207; 7,084,125; and 7,399,845, each of which is hereinincorporated by reference in its entirety.

Representative U.S. patents that teach the preparation of PNA compoundsinclude, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331;and 5,719,262, each of which is herein incorporated by reference.Further teaching of PNA compounds can be found, for example, in Nielsenet al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the disclosure include polynucleotides withphosphorothioate backbones and oligonucleosides with other modifiedbackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as amethylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂—[wherein the nativephosphodiester backbone is represented as —O—P(O)₂—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, thepolynucleotides featured herein have morpholino backbone structures ofthe above-referenced U.S. Pat. No. 5,034,506.

Modifications at the 2′ position can also aid in delivery. For example,modifications at the 2′ position are not located in a polypeptide-codingsequence, i.e., not in a translatable region. Modifications at the 2′position can be located in a 5′UTR, a 3′UTR and/or a tailing region.Modifications at the 2′ position can include one of the following at the2′ position: H (i.e., 2′-deoxy); F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O—, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, the polynucleotidesinclude one of the following at the 2′ position: C₁ to C₁₀ lower alkyl,substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties, or agroup for improving the pharmacodynamic properties, and othersubstituents having similar properties. In some embodiments, themodification includes a 2′-methoxyethoxy (2′-O—˜CH₂CH₂OCH₃, also knownas 2′-O—(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta,1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplarymodification is 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, as described in examples herein below,and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—˜CH₂—O—˜CH₂—N(CH₂)₂, also described in examples herein below. Othermodifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can alsobe made at other positions, particularly the 3′ position of the sugar onthe 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ positionof 5′ terminal nucleotide. Polynucleotides of the disclosure can alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920; the contents of each ofwhich is herein incorporated by reference in their entirety.

In still other embodiments, the polynucleotide is covalently conjugatedto a cell penetrating polypeptide. The cell-penetrating peptide can alsoinclude a signal sequence or a targeting sequence. The conjugates of thedisclosure can be designed to have increased stability; increased celltransfection; and/or altered the biodistribution (e.g., targeted tospecific tissues or cell types).

In some embodiments, the polynucleotides can be conjugated to an agentto enhance delivery. As a non-limiting example, the agent can be amonomer or polymer such as a targeting monomer or a polymer havingtargeting blocks as described in International Publication No.WO2011062965, herein incorporated by reference in its entirety. Inanother non-limiting example, the agent can be a transport agentcovalently coupled to the polynucleotides of the present disclosure(See, e.g., U.S. Pat. Nos. 6,835.393 and 7,374,778, each of which isherein incorporated by reference in its entirety). In yet anothernon-limiting example, the agent can be a membrane barrier transportenhancing agent such as those described in U.S. Pat. Nos. 7,737,108 and8,003,129, each of which is herein incorporated by reference in itsentirety.

In another embodiment, polynucleotides can be conjugated to SMARTTPOLYMER TECHNOLOGY® (PHASERX®, Inc. Seattle, Wash.).

In another aspect, the conjugate can be a peptide that selectivelydirects the nanoparticle to neurons in a tissue or organism. As anon-limiting example, the peptide used can be, but is not limited to,the peptides described in US Patent Publication No US20130129627, hereinincorporated by reference in its entirety.

In yet another aspect, the conjugate can be a peptide that can assist incrossing the blood-brain barrier.

Nanoparticle Formulations

Self-Assembled Nanoparticles

The disclosure also includes pharmaceutical compositions that comprise aformulation of the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide, withself-assembled nanoparticles. Nucleic acid self-assembled nanoparticlesare described in International Patent Application No. PCT/US2014/027077,the contents of which are herein incorporated by reference in itsentirety, such as in paragraphs [000740]-[000743]. Polymer-basedself-assembled nanoparticles are described in International PatentApplication No. PCT/US2014/027077, the contents of which are hereinincorporated by reference in its entirety.

Nanoparticle Mimics

The disclosure includes pharmaceutical compositions that comprise ananoparticle mimic formulation of the polynucleotide described herein,i.e., a polynucleotide comprising an ORF encoding an MCM polypeptide. Insome embodiments, the polynucleotides of the disclosure is beencapsulated within and/or absorbed to a nanoparticle mimic. Ananoparticle mimic can mimic the delivery function organisms orparticles such as, but not limited to, pathogens, viruses, bacteria,fungus, parasites, prions and cells. As a non-limiting example thepolynucleotides of the disclosure can be encapsulated in a non-vironparticle that can mimic the delivery function of a virus (seeInternational Pub. No. WO2012006376 and US Patent Publication No.US20130171241 and US20130195968, the contents of each of which areherein incorporated by reference in its entirety).

Nanotubes

The disclosure includes pharmaceutical compositions that comprise ananotube formulation of the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide. Thepolynucleotides of the disclosure can be attached or otherwise bound toat least one nanotube such as, but not limited to, rosette nanotubes,rosette nanotubes having twin bases with a linker, carbon nanotubesand/or single-walled carbon nanotubes, The polynucleotides can be boundto the nanotubes through forces such as, but not limited to, steric,ionic, covalent and/or other forces. Nanotubes and nanotube formulationscomprising polynucleotides are described in International PatentApplication No. PCT/US2014/027077, the contents of which are hereinincorporated by reference in its entirety.

Inorganic Nanoparticles

The disclosure also includes pharmaceutical compositions that comprise aformulation of the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide, ininorganic nanoparticles. Example methods are provided in U.S. Pat. No.8,257,745, herein incorporated by reference in its entirety. Theinorganic nanoparticles can include, but are not limited to, claysubstances that are water swellable. As a non-limiting example, theinorganic nanoparticle can include synthetic smectite clays that aremade from simple silicates (See, e.g., U.S. Pat. Nos. 5,585,108 and8,257,745 each of which are herein incorporated by reference in theirentirety).

In some embodiments, the inorganic nanoparticles can comprise a core ofthe polynucleotides disclosed herein and a polymer shell. The polymershell can be any of the polymers described herein and are known in theart. In an additional embodiment, the polymer shell can be used toprotect the polynucleotides in the core.

Semi-Conductive and Metallic Nanoparticles

The disclosure also includes pharmaceutical compositions that comprise aformulation of the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide, withwater-dispersible nanoparticles comprising a semiconductive or metallicmaterial (U.S. Pub. No. 20120228565; herein incorporated by reference inits entirety) or formed in a magnetic nanoparticle (U.S. Pub. No.20120265001 and 20120283503; each of which is herein incorporated byreference in its entirety). The water-dispersible nanoparticles can behydrophobic nanoparticles or hydrophilic nanoparticles.

In some embodiments, the semi-conductive and/or metallic nanoparticlescan comprise a core of the polynucleotides disclosed herein and apolymer shell. The polymer shell can be any of the polymers describedherein and are known in the art. In an additional embodiment, thepolymer shell can be used to protect the polynucleotides in the core.

Molded Nanoparticles and Microparticles

The disclosure also includes pharmaceutical compositions that comprise aformulation of the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide, innanoparticles and/or microparticles. These nanoparticles and/ormicroparticles can be molded into any size shape and chemistry. As anexample, the nanoparticles and/or microparticles can be made using thePRINT® technology by LIQUIDA TECHNOLOGIES® (Morrisville, N.C.) (See,e.g., International Pub. No. WO2007024323; the contents of which areherein incorporated by reference in its entirety).

In some embodiments, the molded nanoparticles can comprise a core of thepolynucleotides disclosed herein and a polymer shell. The polymer shellcan be any of the polymers described herein and are known in the art. Inan additional embodiment, the polymer shell can be used to protect thepolynucleotides in the core.

In some embodiments, the polynucleotides of the present disclosure canbe formulated in microparticles. The microparticles can contain a coreof the polynucleotides and a cortex of a biocompatible and/orbiodegradable polymer. As a non-limiting example, the microparticlesthat can be used with the present disclosure can be those described inU.S. Pat. No. 8,460,709, U.S. Patent Publication No. US20130129830 andInternational Patent Publication No WO2013075068, each of which isherein incorporated by reference in its entirety. As anothernon-limiting example, the microparticles can be designed to extend therelease of the polynucleotides of the present disclosure over a desiredperiod of time (see, e.g., extended release of a therapeutic protein inU.S. Patent Publication No. US20130129830, herein incorporated byreference in its entirety).

The microparticle for use with the present disclosure can have adiameter of at least 1 micron to at least 100 microns (e.g., at least 1micron, at least 5 micron, at least 10 micron, at least 15 micron, atleast 20 micron, at least 25 micron, at least 30 micron, at least 35micron, at least 40 micron, at least 45 micron, at least 50 micron, atleast 55 micron, at least 60 micron, at least 65 micron, at least 70micron, at least 75 micron, at least 80 micron, at least 85 micron, atleast 90 micron, at least 95 micron, at least 97 micron, at least 99micron, and at least 100 micron).

NanoJackets and NanoLiposomes

The disclosure also includes pharmaceutical compositions that comprise aformulation of the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide, withNanoJackets and NanoLiposomes by Keystone Nano (State College, Pa.).NanoJackets are made of compounds that are naturally found in the bodyincluding calcium, phosphate and can also include a small amount ofsilicates. Nanojackets can range in size from 5 to 50 nm and can be usedto deliver hydrophilic and hydrophobic compounds such as, but notlimited to, polynucleotides.

NanoLiposomes are made of lipids such as, but not limited to, lipidsthat naturally occur in the body. NanoLiposomes can range in size from60-80 nm and can be used to deliver hydrophilic and hydrophobiccompounds such as, but not limited to, polynucleotides. In one aspect,the polynucleotides disclosed herein are formulated in a NanoLiposomesuch as, but not limited to, Ceramide NanoLiposomes. Cells

The disclosure also includes cells that comprise a polynucleotidedescribed herein, i.e., a polynucleotide comprising an ORF encoding anMCM polypeptide. The polynucleotides of the disclosure can betransfected ex vivo into cells, which are subsequently transplanted intoa subject. As non-limiting examples, the pharmaceutical compositions caninclude red blood cells to deliver modified RNA to liver and myeloidcells, virosomes to deliver modified RNA in virus-like particles (VLPs),and electroporated cells such as, but not limited to, from MAXCYTE®(Gaithersburg, Md.) and from ERYTECH® (Lyon, France) to deliver modifiedRNA. Examples of use of red blood cells, viral particles andelectroporated cells to deliver payloads other than polynucleotides havebeen documented (Godfrin et al., Expert Opin Biol Ther. 2012 12:127-133;Fang et al., Expert Opin Biol Ther. 2012 12:385-389; Hu et al., ProcNatl Acad Sci USA. 2011 108:10980-10985; Lund et al., Pharm Res. 201027:400-420; Huckriede et al., J Liposome Res. 2007; 17:39-47; Cusi, HumVaccin. 2006 2:1-7; de Jonge et al., Gene Ther. 2006 13:400-411; all ofwhich are herein incorporated by reference in its entirety).

The polynucleotides can be delivered in synthetic VLPs synthesized bythe methods described in International Pub No. WO2011085231 andWO2013116656 and US Pub No. 20110171248, the contents of each of whichare herein incorporated by reference in their entireties.

Cell-based formulations of the polynucleotides of the disclosure can beused to ensure cell transfection (e.g., in the cellular carrier), alterthe biodistribution of the polynucleotide (e.g., by targeting the cellcarrier to specific tissues or cell types), and/or increase thetranslation of encoded protein.

A variety of methods are known in the art and suitable for introductionof nucleic acid into a cell, including viral and non-viral mediatedtechniques. Examples of typical non-viral mediated techniques include,but are not limited to, electroporation, calcium phosphate mediatedtransfer, nucleofection, sonoporation, heat shock, magnetofection,liposome mediated transfer, microinjection, microprojectile mediatedtransfer (nanoparticles), cationic polymer mediated transfer(DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like)or cell fusion.

The technique of sonoporation, or cellular sonication, is the use ofsound (e.g., ultrasonic frequencies) for modifying the permeability ofthe cell plasma membrane. Sonoporation methods are known to those in theart and are used to deliver nucleic acids in vivo (Yoon and Park, ExpertOpin Drug Deliv. 2010 7:321-330; Postema and Gilja, Curr PharmBiotechnol. 2007 8:355-361; Newman and Bettinger, Gene Ther. 200714:465-475; all herein incorporated by reference in their entirety).Sonoporation methods are known in the art and are also taught forexample as it relates to bacteria in US Patent Publication 20100196983and as it relates to other cell types in, for example, US PatentPublication 20100009424, each of which are incorporated herein byreference in their entirety.

Electroporation techniques are also well known in the art and are usedto deliver nucleic acids in vivo and clinically (Andre et al., Curr GeneTher. 2010 10:267-280; Chiarella et al., Curr Gene Ther. 201010:281-286; Hojman, Curr Gene Ther. 2010 10:128-138; all hereinincorporated by reference in their entirety). Electroporation devicesare sold by many companies worldwide including, but not limited to BTX®Instruments (Holliston, Mass.) (e.g., the AgilePulse In Vivo System) andInovio (Blue Bell, Pa.) (e.g., Inovio SP-5P intramuscular deliverydevice or the CELLECTRA® 3000 intradermal delivery device). In someembodiments, polynucleotides can be delivered by electroporation.

In some embodiments, the cells are selected from the group consisting ofmammalian cells, bacterial cells, plant, microbial, algal and fungalcells. In some embodiments, the cells are mammalian cells, such as, butnot limited to, human, mouse, rat, goat, horse, rabbit, hamster or cowcells. In a further embodiment, the cells can be from an establishedcell line, including, but not limited to, HeLa, NS0, SP2/0, KEK 293T,Vero, Caco, Caco-2, MDCK, COS-1, COS-7, K562, Jurkat, CHO-K1, DG44,CHOK1SV, CHO-S, Huvec, CV-1, Huh-7, NIH3T3, HEK293, 293, A549, HepG2,IMR-90, MCF-7, U-20S, Per.C6, SF9, SF21 or Chinese Hamster Ovary (CHO)cells.

In certain embodiments, the cells are fungal cells, such as, but notlimited to, Chrysosporium cells, Aspergillus cells, Trichoderma cells,Dictyostelium cells, Candida cells, Saccharomyces cells,Schizosaccharomyces cells, and Penicillium cells.

In certain embodiments, the cells are bacterial cells such as, but notlimited to, E. coli, B. subtilis, or BL21 cells. Primary and secondarycells to be transfected by the methods of the disclosure can be obtainedfrom a variety of tissues and include, but are not limited to, all celltypes that can be maintained in culture. For examples, primary andsecondary cells that can be transfected by the methods of the disclosureinclude, but are not limited to, fibroblasts, keratinocytes, epithelialcells (e.g., mammary epithelial cells, intestinal epithelial cells),endothelial cells, glial cells, neural cells, formed elements of theblood (e.g., lymphocytes, bone marrow cells), muscle cells andprecursors of these somatic cell types. Primary cells can also beobtained from a donor of the same species or from another species (e.g.,mouse, rat, rabbit, cat, dog, pig, cow, bird, sheep, goat, horse).

Minicells

The disclosure also includes minicells that comprise a polynucleotidedescribed herein, i.e., a polynucleotide comprising an ORF encoding anMCM polypeptide. In one aspect, the polynucleotides can be formulated inbacterial minicells. As a non-limiting example, bacterial minicells canbe those described in International Publication No. WO2013088250 or USPatent Publication No. US20130177499, the contents of each of which areherein incorporated by reference in its entirety.

Micro-Organs

The disclosure also includes micro-organs containing a polynucleotidedescribed herein, i.e., a polynucleotide comprising an ORF encoding anMCM polypeptide. The polynucleotides can be contained in a micro-organthat can then express an encoded polypeptide of interest in along-lasting therapeutic formulation. Micro-organs and formulationsthereof are described in International Patent Application No.PCT/US2014/027077, the contents of which are herein incorporated byreference in its entirety.

Exosomes

The disclosure also includes pharmaceutical compositions that comprise aformulation of the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide, inexosomes. The exosomes can be loaded with at least one polynucleotideand delivered to cells, tissues and/or organisms. As a non-limitingexample, the polynucleotides can be loaded in the exosomes described inInternational Publication No. WO2013084000, herein incorporated byreference in its entirety.

Pseudovirions

The disclosure also includes pharmaceutical compositions that comprise aformulation of the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide, inPseudovirions (e.g., pseudo-virions). As a non-limiting example, thepseudovirions can be those developed and/or are described by AuraBiosciences (Cambridge, Mass.). In one aspect, the pseudovirion can bedeveloped to deliver drugs to keratinocytes and basal membranes (Seee.g., US Patent Publication Nos. US20130012450, US20130012566,US21030012426 and US20120207840 and International Publication No.WO2013009717, each of which is herein incorporated by reference in itsentirety).

In some embodiments, the pseudovirion used for delivering thepolynucleotides of the present disclosure can be derived from virusessuch as, but not limited to, herpes and papillomaviruses (See, e.g., USPatent Publication Nos. US Patent Publication Nos. US20130012450,US20130012566, US21030012426 and US20120207840 and InternationalPublication No. WO2013009717, each of which is herein incorporated byreference in its entirety; and Ma et al. HPV pseudovirions as DNAdelivery vehicles. Ther Deliv. 2011: 2(4): 427-430; Kines et al. Theinitial steps leading to papillomavirus infection occur on the basementmembrane prior to cell surface binding. PNAS 2009:106(48), 20458-20463;Roberts et al. Genital transmission of HPV in a mouse model ispotentiated by nonoxynol-9 and inhibited by carrageenan. NatureMedicine. 2007:13(7) 857-861; Gordon et al., Targeting the VaginalMucosa with Human Papillomavirus Pseudovirion Vaccines delivering SIVDNA. J Immunol. 2012 188(2) 714-723; Cuburu et al., Intravaginalimmunization with HPV vectors induces tissue-resident CD8+ T cellresponses. The Journal of Clinical Investigation. 2012: 122(12)4606-4620; Hung et al., Ovarian Cancer Gene Therapy Using HPV-16Pseudovirion Carrying the HSV-tk Gene. PLoS ONE. 2012: 7(7) e40983;Johnson et al., Role of Heparan Sulfate in Attachment to and Infectionof the Murine Female Genital Tract by Human Papillomavirus. J Virology.2009: 83(5) 2067-2074; each of which is herein incorporated by referencein its entirety).

The pseudovirion can be a virus-like particle (VLP) prepared by themethods described in US Patent Publication No. US20120015899 andUS20130177587 and International Patent Publication No. WO2010047839WO2013116656, WO2013106525 and WO2013122262, the contents of each ofwhich is herein incorporated by reference in its entirety. In oneaspect, the VLP can be, but is not limited to, bacteriophages MS, QP,R₁₇, fr, GA, Sp, MI, I, MXI, NL95, AP205, f2, PP7, and the plant virusesTurnip crinkle virus (TCV), Tomato bushy stunt virus (TBSV), Southernbean mosaic virus (SBMV) and members of the genus Bromovirus includingBroad bean mottle virus, Brome mosaic virus, Cassia yellow blotch virus,Cowpea chlorotic mottle virus (CCMV), Melandrium yellow fleck virus, andSpring beauty latent virus. In another aspect, the VLP can be derivedfrom the influenza virus as described in US Patent Publication No.US20130177587 or U.S. Pat. No. 8,506,967, the contents of each of whichare herein incorporated by reference in its entirety. In yet anotheraspect, the VLP can comprise a B7-1 and/or B7-2 molecule anchored to alipid membrane or the exterior of the particle such as described inInternational Patent Publication No. WO2013116656, the contents of whichare herein incorporated by reference in its entirety. In one aspect, theVLP can be derived from norovirus, rotavirus recombinant VP6 protein ordouble layered VP2/VP6 such as the VLP described in International PatentPublication No. WO2012049366, the contents of which are hereinincorporated by reference in its entirety.

The pseudovirion can be a human papilloma virus-like particle such as,but not limited to, those described in International Publication No.WO2010120266 and US Patent Publication No. US20120171290, each of whichis herein incorporated by reference in its entirety and Ma et al. HPVpseudovirions as DNA delivery vehicles. Ther Deliv. 2011: 2(4): 427-430;Kines et al. The initial steps leading to papillomavirus infection occuron the basement membrane prior to cell surface binding. PNAS2009:106(48), 20458-20463; Roberts et al. Genital transmission of HPV ina mouse model is potentiated by nonoxynol-9 and inhibited bycarrageenan. Nature Medicine. 2007:13(7) 857-861; Gordon et al.,Targeting the Vaginal Mucosa with Human Papillomavirus PseudovirionVaccines delivering SIV DNA. J Immunol. 2012 188(2) 714-723; Cuburu etal., Intravaginal immunization with HPV vectors induces tissue-residentCD8+ T cell responses. The Journal of Clinical Investigation. 2012:122(12) 4606-4620; Hung et al., Ovarian Cancer Gene Therapy Using HPV-16Pseudovirion Carrying the HSV-tk Gene. PLoS ONE. 2012: 7(7) e40983;Johnson et al., Role of Heparan Sulfate in Attachment to and Infectionof the Murine Female Genital Tract by Human Papillomavirus. J Virology.2009: 83(5) 2067-2074; each of which is herein incorporated by referencein its entirety.

In one aspect, the pseudovirions can be virion derived nanoparticlessuch as, but not limited to, those described in US Patent PublicationNo. US20130116408 and US20130115247, each of which is hereinincorporated by reference in their entirety. The virion derivednanoparticles can be made by the methods described in US PatentPublication No. US20130116408 and US20130115247 or International PatentPublication No. WO2013119877, each of which is herein incorporated byreference in their entirety.

In some embodiments, the virus-like particle (VLP) can be aself-assembled particle. Non-limiting examples of self-assembled VLPsand methods of making the self-assembled VLPs are described inInternational Patent Publication No. WO2013122262, the contents of whichare herein incorporated by reference in its entirety.

Silk-Based Delivery

The disclosure also includes pharmaceutical compositions that areformulated for silk-based delivery of the polynucleotides describedherein, i.e., a polynucleotide comprising an ORF encoding an MCMpolypeptide. In some embodiments, the polynucleotides can be formulatedin a sustained release silk-based delivery system. The silk-baseddelivery system can be formed by contacting a silk fibroin solution witha therapeutic agent such as, but not limited to, the polynucleotidesdescribed herein and/or known in the art. As a non-limiting example, thesustained release silk-based delivery system that can be used in thepresent disclosure and methods of making such system are described in USPatent Publication No. US20130177611, the contents of which are hereinincorporated by reference in its entirety.

Microparticles

The disclosure includes pharmaceutical compositions that comprise amicroparticle formulation of the polynucleotide described herein, i.e.,a polynucleotide comprising an ORF encoding an MCM polypeptide. In someembodiments, formulations comprising polynucleotides can comprisemicroparticles. The microparticles can comprise a polymer describedherein and/or known in the art such as, but not limited to,poly(α-hydroxy acid), a polyhydroxy butyric acid, a polycaprolactone, apolyorthoester and a polyanhydride. The microparticle can have adsorbentsurfaces to adsorb biologically active molecules such aspolynucleotides. As a non-limiting example microparticles for use withthe present disclosure and methods of making microparticles aredescribed in US Patent Publication No. US2013195923 and US20130195898and U.S. Pat. Nos. 8,309,139 and 8,206,749, the contents of each ofwhich are herein incorporated by reference in its entirety.

In another embodiment, the formulation can be a microemulsion comprisingmicroparticles and polynucleotides. As a non-limiting example,microemulsions comprising microparticles are described in US PatentPublication No. US2013195923 and US20130195898 and U.S. Pat. Nos.8,309,139 and 8,206,749, the contents of each of which are hereinincorporated by reference in its entirety.

Microvesicles

The disclosure includes pharmaceutical compositions that comprise amicrovesicle-based formulation of the polynucleotide described herein,i.e., a polynucleotide comprising an ORF encoding an MCM polypeptide. Insome embodiments, polynucleotides can be formulated in microvesicles.Non-limiting examples of microvesicles include those described in USPatent Publication No. US20130209544, the contents of which are hereinincorporated by reference in its entirety.

In some embodiments, the microvesicle is an ARRDC1-mediatedmicrovesicles (ARMMs). Non-limiting examples of ARMMs and methods ofmaking ARMMs are described in International Patent Publication No.WO2013119602, the contents of which are herein incorporated by referencein its entirety.

Interpolyelectrolyte Complexes

The disclosure also includes pharmaceutical compositions that comprise aformulation of the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide, in aninterpolyelectrolyte complex. Interpolyelectrolyte complexes are formedwhen charge-dynamic polymers are complexed with one or more anionicmolecules. Non-limiting examples of charge-dynamic polymers andinterpolyelectrolyte complexes and methods of makinginterpolyelectrolyte complexes are described in U.S. Pat. No. 8,524,368,the contents of which is herein incorporated by reference in itsentirety.

Crystalline Polymeric Systems

The disclosure also includes pharmaceutical compositions that comprise aformulation of the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide, incrystalline polymeric systems. Crystalline polymeric systems arepolymers with crystalline moieties and/or terminal units comprisingcrystalline moieties. Non-limiting examples of polymers with crystallinemoieties and/or terminal units comprising crystalline moieties termed“CYC polymers,” crystalline polymer systems and methods of making suchpolymers and systems are described in U.S. Pat. No. 8,524,259, thecontents of which are herein incorporated by reference in its entirety.

Cryoprotectants for mRNA

The disclosure also includes pharmaceutical compositions that comprise aformulation of the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide, withcyroprotectants. As used herein, there term “cryoprotectant” refers toone or more agent that when combined with a given substance, helps toreduce or eliminate damage to that substance that occurs upon freezing.In some embodiments, cryoprotectants are combined with polynucleotidesin order to stabilize them during freezing. Frozen storage of mRNAbetween −20° C. and −80° C. can be advantageous for long term (e.g., 36months) stability of polynucleotide. In some embodiments,cryoprotectants are included in polynucleotide formulations to stabilizepolynucleotide through freeze/thaw cycles and under frozen storageconditions. Cryoprotectants of the present disclosure can include, butare not limited to sucrose, trehalose, lactose, glycerol, dextrose,raffinose and/or mannitol. Trehalose is listed by the Food and DrugAdministration as being generally regarded as safe (GRAS) and iscommonly used in commercial pharmaceutical formulations.

Bulking Agents

The disclosure also includes pharmaceutical compositions that comprise aformulation of the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide, withbulking agents. As used herein, the term “bulking agent” refers to oneor more agents included in formulations to impart a desired consistencyto the formulation and/or stabilization of formulation components. Insome embodiments, bulking agents are included in lyophilizedpolynucleotide formulations to yield a “pharmaceutically elegant” cake,stabilizing the lyophilized polynucleotides during long term (e.g., 36month) storage. Bulking agents of the present disclosure can include,but are not limited to sucrose, trehalose, mannitol, glycine, lactoseand/or raffinose. In some embodiments, combinations of cryoprotectantsand bulking agents (for example, sucrose/glycine or trehalose/mannitol)can be included to both stabilize polynucleotides during freezing andprovide a bulking agent for lyophilization.

Non-limiting examples of formulations and methods for formulating thepolynucleotides of the present disclosure are also provided inInternational Publication No WO2013090648 filed Dec. 14, 2012, thecontents of which are incorporated herein by reference in theirentirety.

Inactive Ingredients

The disclosure also includes pharmaceutical compositions that comprise aformulation of the polynucleotide described herein, i.e., apolynucleotide comprising an ORF encoding an MCM polypeptide, with atleast one excipient that is an inactive ingredient. As used herein, theterm “inactive ingredient” refers to one or more inactive agentsincluded in formulations. In some embodiments, all, none or some of theinactive ingredients that can be used in the formulations of the presentdisclosure can be approved by the US Food and Drug Administration (FDA).A non-exhaustive list of inactive ingredients and the routes ofadministration the inactive ingredients can be formulated in aredescribed in International Application No. PCT/US2014/027077.

VII. METHOD OF USE OF POLYNUCLEOTIDES

The polynucleotides of the present disclosure (i.e., a polynucleotidecomprising an ORF encoding an MCM polypeptide) can be administered byany route that results in a therapeutically effective outcome. Theseinclude, but are not limited to enteral (into the intestine),gastroenteral, epidural (into the dura matter), oral (by way of themouth), transdermal, peridural, intracerebral (into the cerebrum),intracerebroventricular (into the cerebral ventricles), epicutaneous(application onto the skin), intradermal, (into the skin itself),subcutaneous (under the skin), nasal administration (through the nose),intravenous (into a vein), intravenous bolus, intravenous drip,intraarterial (into an artery), intramuscular (into a muscle),intracardiac (into the heart), intraosseous infusion (into the bonemarrow), intrathecal (into the spinal canal), intraperitoneal, (infusionor injection into the peritoneum), intravesical infusion, intravitreal,(through the eye), intracavernous injection (into a pathologic cavity)intracavitary (into the base of the penis), intravaginal administration,intrauterine, extra-amniotic administration, transdermal (diffusionthrough the intact skin for systemic distribution), transmucosal(diffusion through a mucous membrane), transvaginal, insufflation(snorting), sublingual, sublabial, enema, eye drops (onto theconjunctiva), in ear drops, auricular (in or by way of the ear), buccal(directed toward the cheek), conjunctival, cutaneous, dental (to a toothor teeth), electro-osmosis, endocervical, endosinusial, endotracheal,extracorporeal, hemodialysis, infiltration, interstitial,intra-abdominal, intra-amniotic, intra-articular, intrabiliary,intrabronchial, intrabursal, intracartilaginous (within a cartilage),intracaudal (within the cauda equine), intracistemal (within the cistemamagna cerebellomedularis), intracorneal (within the cornea), dentalintracornal, intracoronary (within the coronary arteries), intracorporuscavernosum (within the dilatable spaces of the corporus cavernosa of thepenis), intradiscal (within a disc), intraductal (within a duct of agland), intraduodenal (within the duodenum), intradural (within orbeneath the dura), intraepidermal (to the epidermis), intraesophageal(to the esophagus), intragastric (within the stomach), intragingival(within the gingivae), intraileal (within the distal portion of thesmall intestine), intralesional (within or introduced directly to alocalized lesion), intraluminal (within a lumen of a tube),intralymphatic (within the lymph), intramedullary (within the marrowcavity of a bone), intrameningeal (within the meninges), intraocular(within the eye), intraovarian (within the ovary), intrapericardial(within the pericardium), intrapleural (within the pleura),intraprostatic (within the prostate gland), intrapulmonary (within thelungs or its bronchi), intrasinal (within the nasal or periorbitalsinuses), intraspinal (within the vertebral column), intrasynovial(within the synovial cavity of a joint), intratendinous (within atendon), intratesticular (within the testicle), intrathecal (within thecerebrospinal fluid at any level of the cerebrospinal axis),intrathoracic (within the thorax), intratubular (within the tubules ofan organ), intratumor (within a tumor), intratympanic (within the aurusmedia), intravascular (within a vessel or vessels), intraventricular(within a ventricle), iontophoresis (by means of electric current whereions of soluble salts migrate into the tissues of the body), irrigation(to bathe or flush open wounds or body cavities), laryngeal (directlyupon the larynx), nasogastric (through the nose and into the stomach),occlusive dressing technique (topical route administration that is thencovered by a dressing that occludes the area), ophthalmic (to theexternal eye), oropharyngeal (directly to the mouth and pharynx),parenteral, percutaneous, periarticular, peridural, perineural,periodontal, rectal, respiratory (within the respiratory tract byinhaling orally or nasally for local or systemic effect), retrobulbar(behind the pons or behind the eyeball), intramyocardial (entering themyocardium), soft tissue, subarachnoid, subconjunctival, submucosal,topical, transplacental (through or across the placenta), transtracheal(through the wall of the trachea), transtympanic (across or through thetympanic cavity), ureteral (to the ureter), urethral (to the urethra),vaginal, caudal block, diagnostic, nerve block, biliary perfusion,cardiac perfusion, photopheresis or spinal. In specific embodiments,compositions can be administered in a way that allows them cross theblood-brain barrier, vascular barrier, or other epithelial barrier. Insome embodiments, a formulation for a route of administration caninclude at least one inactive ingredient.

The polynucleotides of the present disclosure can be delivered to a cellnaked. As used herein in, “naked” refers to delivering polynucleotidesfree from agents that promote transfection. For example, thepolynucleotides delivered to the cell can contain no modifications. Thenaked polynucleotides can be delivered to the cell using routes ofadministration known in the art and described herein.

The polynucleotides of the present disclosure can be formulated, usingthe methods described herein. The formulations can containpolynucleotides that can be modified and/or unmodified. The formulationscan further include, but are not limited to, cell penetration agents, apharmaceutically acceptable carrier, a delivery agent, a bioerodible orbiocompatible polymer, a solvent, and a sustained-release deliverydepot. The formulated polynucleotides can be delivered to the cell usingroutes of administration known in the art and described herein.

The compositions can also be formulated for direct delivery to an organor tissue in any of several ways in the art including, but not limitedto, direct soaking or bathing, via a catheter, by gels, powder,ointments, creams, gels, lotions, and/or drops, by using substrates suchas fabric or biodegradable materials coated or impregnated with thecompositions, and the like.

Parenteral and Injectable Administration

The present disclosure encompasses the delivery of polynucleotides ofthe disclosure (i.e., a polynucleotide comprising an ORF encoding an MCMpolypeptide) in forms suitable for parenteral and injectableadministration. Liquid dosage forms for parenteral administrationinclude, but are not limited to, pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups, and/or elixirs. Inaddition to active ingredients, liquid dosage forms can comprise inertdiluents commonly used in the art such as, for example, water or othersolvents, solubilizing agents and emulsifiers such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, oral compositions can includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, and/or perfuming agents. In certain embodimentsfor parenteral administration, compositions are mixed with solubilizingagents such as CREMOPHOR®, alcohols, oils, modified oils, glycols,polysorbates, cyclodextrins, polymers, and/or combinations thereof.

A pharmaceutical composition for parenteral administration can compriseat least one inactive ingredient. Any or none of the inactiveingredients used can have been approved by the US Food and DrugAdministration (FDA). A non-exhaustive list of inactive ingredients foruse in pharmaceutical compositions for parenteral administrationincludes hydrochloric acid, mannitol, nitrogen, sodium acetate, sodiumchloride and sodium hydroxide.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions can be formulated according to the known artusing suitable dispersing agents, wetting agents, and/or suspendingagents. Sterile injectable preparations can be sterile injectablesolutions, suspensions, and/or emulsions in nontoxic parenterallyacceptable diluents and/or solvents, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution, U.S.P., and isotonic sodiumchloride solution. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. Fatty acids suchas oleic acid can be used in the preparation of injectables. The sterileformulation can also comprise adjuvants such as local anesthetics,preservatives and buffering agents.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions that can bedissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Injectable formulations can be for direct injection into a region of atissue, organ and/or subject. As a non-limiting example, a tissue, organand/or subject can be directly injected a formulation by intramyocardialinjection into the ischemic region. (See, e.g., Zangi et al. NatureBiotechnology 2013; the contents of which are herein incorporated byreference in its entirety).

In order to prolong the effect of an active ingredient, it is oftendesirable to slow the absorption of the active ingredient fromsubcutaneous or intramuscular injection. This can be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the drug then dependsupon its rate of dissolution which, in turn, can depend upon crystalsize and crystalline form. Alternatively, delayed absorption of aparenterally administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle. Injectable depot forms are madeby forming microencapsule matrices of the drug in biodegradable polymerssuch as polylactide-polyglycolide. Depending upon the ratio of drug topolymer and the nature of the particular polymer employed, the rate ofdrug release can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissues.

Therapeutic Use

The polynucleotides of the present disclosure are used in thepreparation, manufacture and therapeutic use of polynucleotide moleculescomprising an mRNA encoding a methylmalonyl-CoA mutase (MCM)polypeptide. In some embodiments, the polynucleotides of the presentdisclosure can be used to treat and/or prevent MCM-related diseases,disorders or conditions. Typically, but not exclusively, thepolynucleotides of the present disclosure can be used to treat and/orprevent methylmalonic acidemia. In some embodiments, the nucleotides areused in methods for reducing the levels of methylmalonic acid in asubject in need thereof. For instance, one aspect of the disclosureprovides a method of alleviating the symptoms of methylmalonic acidemiain a subject via the administration of a composition comprising apolynucleotide encoding MCM to that subject.

In some embodiments, the polynucleotides of the present disclosure areused to reduce the level of a metabolite associated with methylmalonicacidemia, the method comprising administering to the subject aneffective amount of a polynucleotide encoding an MCM polypeptide. Insome embodiments, the administration of an effective amount of apolynucleotide reduces the levels of a biomarker of methylmalonicacidemia such as methylmalonic acid, propionyl-carnitine,acetyl-carnitine, propionyl-CoA, D-methylmalonyl-CoA,L-methylmalonyl-CoA, or a combination thereof in a subject. In someembodiments, the administration of the polynucleotide results inreduction in the level of one or more biomarkers of methylmalonicacidemia within a short period of time after administration of thepolynucleotide.

In some embodiments, the administration of the polynucleotide results inexpression of methylmalonyl-CoA mutase in cells of the subject. In someembodiments, administering the polynucleotide results in an increase ofMCM enzymatic activity in the subject. For example, in some embodiments,the polynucleotides of the present disclosure are used in methods ofadministering a composition comprising an mRNA encoding MCM to asubject, wherein the method results in an increase of MCM enzymaticactivity in at least some cells of a subject. In some embodiments, theadministration of a composition comprising an mRNA encoding MCM to asubject results in an increase of MCM enzymatic activity in cells ofsubject by at least 10%, at least 25%, at least 50%, at least 75%, atleast 100%, or by more than 100%. In some embodiments, theadministration of a composition comprising an mRNA encoding MCM to asubject results in an increase of MCM enzymatic activity in cellssubject to a level at least 10%, at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, or to 100% or more of the activity level expected in a normalsubject, e.g., a normal human not suffering from MMA. In someembodiments, the administration of the polynucleotide results inexpression of methylmalonyl-CoA mutase in at least some of the cells ofa subject that persists for a period of time sufficient to allowsignificant methylmalonyl-CoA metabolism to occur.

In some embodiments, the expression of the encoded polypeptide isincreased. In some embodiments, the IVT polynucleotide increases MCMexpression levels in cells when introduced into those cells, e.g., by20-50%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, or at least 50%.

In some embodiments, the method or use comprises administering apolynucleotide, e.g., mRNA, comprising an ORF having significantsequence similarity to a polynucleotide selected from the group of SEQID NOs: 1-207, 732-765, and 772, wherein the ORF encodes an MCMpolypeptide. Other aspects of the present disclosure relate totransplantation of cells containing polynucleotides to a mammaliansubject. Administration of cells to mammalian subjects is known to thoseof ordinary skill in the art, and includes, but is not limited to, localimplantation (e.g., topical or subcutaneous administration), organdelivery or systemic injection (e.g., intravenous injection orinhalation), and the formulation of cells in pharmaceutically acceptablecarriers. Such compositions containing polynucleotides can be formulatedfor administration intramuscularly, transarterially, intraperitoneally,intravenously, intranasally, subcutaneously, endoscopically,transdermally, or intrathecally.

In some embodiments, the composition can be formulated for extendedrelease.

In some embodiments, the present methods are able to catalyze theconversion of at least 0.1%, 0.5%, 1%, 2%, 2.5%, 5%, 10%, 20%, 25%, 30%,35% 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or more ofL-methylmalonyl-CoA to succinyl-CoA. In some embodiments, a the methodsare able to catalyze the conversion of a range of from 0.1% to 5%, 5% to25%, 10% to 25%, 10% to 30%, 20% to 40%, 10% to 50%, 20% to 50%, 10% to75%, 20% to 75%, 30% to 75%, 30% to 85%, or 25% to 100% ofL-methylmalonyl-CoA to succinyl-CoA.

In some embodiments, the methods achieve at least 0.1%, 0.5%, 1%, 2%,2.5%, 5%, 10%, 20%, 25%, 30%, 35% 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85% or more of the enzymatic activity sufficient for thesynthesis of AdoCbl. In some embodiments, the methods achieve a range offrom 0.1% to 5%, 5% to 25%, 10% to 25%, 10% to 30%, 20% to 40%, 10% to50%, 20% to 50%, 10% to 75%, 20% to 75%, 30% to 75%, 30% to 85%, or 25%to 100% of the enzymatic activity sufficient for the synthesis ofAdenosylcobalamin (AdoCbl).

In some embodiments, the methods achieve sufficient enzymatic activityto synthesize at least 0.1%, 0.5%, 1%, 2%, 2.5%, 5%, 10%, 20%, 25%, 30%,35% 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or more of theactive form of Adenosylcobalamin (AdoCbl), found in healthy individuals.In some embodiments, the methods achieve sufficient enzymatic activityto synthesize a range of from 0.1% to 5%, 5% to 25%, 10% to 25%, 10% to30%, 20% to 40%, 10% to 50%, 20% to 50%, 10% to 75%, 20% to 75%, 30% to75%, 30% to 85%, or 25% to 100% of the active form of AdoCbl found inhealthy individuals.

In some embodiments, the methods involve the administration of apolynucleotide in combination with another therapy to a patient in needthereof. Such methods can involve administering a polynucleotide priorto, concurrent with, or subsequent to administration of the additionaltherapy. In some embodiments, such methods have an additive orsynergistic effect. In some embodiments, presented herein is a methodfor treating MMA, comprising administering to a patient in need thereofan effective amount of a polynucleotide (e.g., mRNA) encoding an MCMpolypeptide and an effective amount of another therapy. Examples of suchother therapies include, but are not limited to, cobalamin supplements,camitine supplements and antibiotics. In another specific embodiment,presented herein is a method for treating MMA, comprising administeringto a patient in need thereof an effective amount of a polynucleotide(e.g., mRNA) encoding an MCM polypeptide and maintaining a low-proteindiet.

In some embodiments, the concentration of methylmalonic acid inbiological specimens (e.g., blood, plasma, serum, cerebral spinal fluid,urine, or any other biofluids) of a patient is monitored before, duringand/or after a course of treatment involving the administration of apolynucleotide (e.g., mRNA) encoding an MCM polypeptide or apharmaceutical composition thereof to the patient. In some embodiments,the concentration of methylcitrate in biological specimens (e.g., urine,blood, plasma, serum, cerebral spinal fluid, or any other biofluids) ofa patient is monitored before, during and/or after a course of treatmentinvolving the administration of a polynucleotide (e.g., mRNA) encodingan MCM polypeptide or a pharmaceutical composition thereof to thepatient. In some embodiments, the concentration of propionylcarnitine inbiological specimens (e.g., blood, plasma, serum, cerebral spinal fluid,urine, or any other biofluids) of a patient is monitored before, duringand/or after a course of treatment involving the administration of apolynucleotide (e.g., mRNA) encoding an MCM polypeptide or apharmaceutical composition thereof to the patient. In some embodiments,erythrocyte odd long-chain fatty acids (OLCFAs) levels are monitoredbefore, during and/or after a course of treatment involving theadministration of a polynucleotide (e.g., mRNA) encoding an MCMpolypeptide or a pharmaceutical composition thereof to a patient. Insome embodiments, the urinary urea:methylmalonic acid ratio is monitoredbefore, during and/or after a course of treatment involving theadministration of a polynucleotide (e.g., mRNA) encoding an MCMpolypeptide or a pharmaceutical composition thereof to a patient. Thedosage, frequency and/or length of administration of a polynucleotide(e.g., mRNA) encoding an MCM polypeptide or a pharmaceutical compositionthereof to a patient may be modified as a result of the concentration ofmethylmalonic acid, methylcitrate, or propionylcarnitine, erythrocyteodd long-chain fatty acids (OLCFAs) levels, or the urinaryurea:methylmalonic acid ratio. Alternatively, changes in one or more ofthese monitoring parameters (e.g., concentration of methylmalonic acid,methylcitrate, or propionylcarnitine, erythrocyte odd long-chain fattyacids (OLCFAs) levels, or the urinary urea:methylmalonic acid ratio)might indicate that the course of treatment involving the administrationof the polynucleotide (e.g., mRNA) encoding an MCM polypeptide orpharmaceutical composition thereof is effective in treating MMA.

In a specific embodiment, presented herein is a method for treating MMA,comprising: (a) administering to a patient in need thereof one or moredoses of a polynucleotide (e.g., mRNA) encoding an MCM polypeptide or apharmaceutical composition thereof; and (b) monitoring the concentrationof methylmalonic acid, methylcitrate, or propionylcarnitine (e.g.,detected in biological specimens such as plasma, serum, cerebral spinalfluid, urine, or other biofluids), erythrocyte odd long-chain fattyacids (OLCFAs) levels, or the urinary urea:methylmalonic acid ratiobefore and/or after step (a). In some embodiments, step (b) comprisesmonitoring the concentration of methylmalonic acid. In some embodiments,step (b) comprises monitoring the concentration of methylmalonic acid,methylcitrate and/or propionylcarnitine. In some embodiments, themonitoring step (b) is carried out before and/or after a certain numberof doses (e.g., 1, 2, 4, 6, 8, 10, 12, 14, 15, or 20 doses, or moredoses; or 2 to 4, 2 to 8, 2 to 20 or 2 to 30 doses) or after a certaintime period (e.g., 1, 2, 3, 4, 5, 6, or 7 days; or 1, 2, 3, 4, 5, 10,15, 20, 30, 40, 45, 48, or 50 weeks) of administering the polynucleotide(e.g., mRNA) encoding an MCM polypeptide. In some embodiments, one ormore of these monitoring parameters are detected prior to administrationof the polynucleotide (e.g., mRNA) encoding an MCM polypeptide orpharmaceutical composition thereof. In some embodiments, a decrease inthe concentration of methylmalonic acid, methylcitrate, orpropionylcamitine, or a decrease in erythrocyte odd long-chain fattyacids (OLCFAs) levels following administration of the polynucleotide orpharmaceutical composition thereof indicates that the course oftreatment is effective for treating MMA. In some embodiments, anincrease in the urinary urea:methylmalonic acid ratio followingadministration of the polynucleotide (e.g., mRNA) encoding an MCMpolypeptide or pharmaceutical composition thereof indicates that thecourse of treatment is effective for treating MMA.

The concentration of methylmalonic acid, methylcitrate, orpropionylcamitine, erythrocyte odd long-chain fatty acids (OLCFAs)levels, or the urinary urea:methylmalonic acid ratio of a patient may bedetected by any technique known to one of skill in the art. In someembodiments, the method for detecting the concentration of methylmalonicacid, methylcitrate, or propionylcarnitine in a patient involvesobtaining a tissue or fluid sample from the patient and detecting theconcentration of methylmalonic acid, methylcitrate, propionylcarnitineor urea in the biological sample (e.g., from plasma serum sample,cerebral spinal fluid, urine, or other biofluids) that has beensubjected to certain types of treatment (e.g., centrifugation) anddetection by use of, e.g., standard gas chromatography/mass spectroscopy(GC/MS) stable-isotope dilution methods, positive chemical ionizationgas chromatography mass spectrometry (CI GC-MS) spectroscopic techniques(e.g., UV spectroscopy) or high pressure liquid chromatography (HPLC).

In some embodiments, the methods for treating MMA provided hereinalleviate or manage one, two or more symptoms associated with MMA.Alleviating or managing one, two or more symptoms of MMA may be used asa clinical endpoint for efficacy of a polynucleotide for treating MMA.In some embodiments, the methods for treating MMA provided herein reducethe duration and/or severity of one or more symptoms associated withMMA. In some embodiments, the methods for treating MMA provided hereininhibit the onset, progression and/or recurrence of one or more symptomsassociated with MMA. In some embodiments, the methods for treating MMAprovided herein reduce the number of symptoms associated with MMA. Insome embodiments, the methods for treating MMA provided herein inhibitor reduce the progression of one or more symptoms associated therewith.

Symptoms associated with MMA include, but are not limited to: apnea,hyperammonemia, metabolic acidosis, lethargy, vomiting, dehydration,hypotonia, hypoglycemia, repeated yeast infections, renal impairment,mental retardation, developmental delays, seizures, movement disorders,progressive encephalopathy, facial dysmorphism (e.g., high forehead,broad nasal bridge, epicanthal folds, long smooth philtrum, ortriangular mouth), stroke, skin lesions (e.g., moniliasis), occasionalhepatomegaly, acute onset of choreoathetosis, dystonia, dysphagia,dysarthria, growth problems (e.g., growth failure), kidney disease orfailure, tissue damage, feeding problems, cognitive disabilities,metabolic attacks triggered by common infections and reduced glomerularfiltration rate (GFR).

In some embodiments, the methods for treating MMA provided herein reduceor eliminate one, two, or more of the following: metabolic acidosis,developmental delays, movement disorders, metabolic decompensationepisodes (e.g., frequency and/or numbers of episodes), skin lesions,hypotonia, seizures, and renal impairment, associated with MMA. In someembodiments, the methods for treating MMA provided herein improve renalfunction, development, cognitive ability and movement in a patientdiagnosed with MMA.

In some embodiments, the methods for treating MMA provided herein reducehospitalization (e.g., the frequency or duration of hospitalization) ofa patient diagnosed with MMA. In some embodiments, the methods fortreating MMA provided herein reduce hospitalization length of a patientdiagnosed with MMA. In some embodiments, the methods for treating MMAprovided herein decrease the hospitalization rate.

In some embodiments, the methods provided herein increase the survivalof a patient diagnosed with MMA. In some embodiments, the methods fortreating MMA provided herein reduce the mortality of subjects diagnosedwith MMA. In some embodiments, the methods for treating MMA providedherein increase symptom-free survival of MMA patients. In someembodiments, the methods for treating MMA provided herein do not cureMMA in patients, but prevent the progression or worsening of thedisease. In some embodiments, the methods for treating MMA providedherein enhance or improve the therapeutic effect of another therapy.

In some embodiments, the methods for treating MMA provided herein reducethe concentration of plasma methylmalonic acid in a subject by at leastabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,80%, 85%, 90%, 95%, or 100%, or in a range of from 5% to 50%, 10% to50%, 20% to 50%, 20% to 75%, 25% to 75%, 25% to 90% or 10% to 99%relative to the respective concentration prior to administration of apolynucleotide (e.g., mRNA) encoding an MCM polypeptide, as assessed bymethods well known in the art or described herein. In some embodiments,the methods for treating MMA provided herein reduce the concentration ofurinary methylmalonic acid in a subject by at least about 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 80%, 85%, 90%, 95%, or100%, or in a range of from 5% to 50%, 10% to 50%, 20% to 50%, 20% to75%, 25% to 75%, 25% to 90% or 10% to 99% relative to the respectiveconcentration prior to administration of a polynucleotide (e.g., mRNA)encoding an MCM polypeptide, as assessed by methods well known in theart or described herein.

In some embodiments, the methods for treating MMA provided herein reducethe concentration of a metabolite of methylmalonic acid in a subject byat least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 80%, 85%, 90%, 95%, or 100%, or in a range of from 5% to 50%,10% to 50%, 20% to 50%, 20% to 75%, 25% to 75%, 25% to 90% or 10% to 99%relative to the respective concentration prior to administration of apolynucleotide (e.g., mRNA) encoding an MCM polypeptide, as assessed bymethods well known in the art or described herein. In some embodiments,the methods for treating MMA provided herein reduce the concentration ofplasma propionylcamitine in a subject by at least about 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 80%, 85%, 90%, 95%, or100%, or in a range of from 5% to 50%, 10% to 50%, 20% to 50%, 20% to75%, 25% to 75%, 25% to 90% or 10% to 99% relative to the respectiveconcentration prior to administration of a polynucleotide (e.g., mRNA)encoding an MCM polypeptide, as assessed by methods well known in theart or described herein. In some embodiments, the methods for treatingMMA provided herein reduce the concentration of urinary methylcitrate ina subject by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 80%, 85%, 90%, 95%, or 100%, or in a range of from10% to 50%, 20% to 50%, 20% to 75%, 25% to 75%, 25% to 90% or 10% to 99%relative to the respective concentration prior to administration of apolynucleotide (e.g., mRNA) encoding an MCM polypeptide, as assessed bymethods well known in the art or described herein.

In some embodiments, the methods for treating MMA provided herein reducethe erythrocyte odd-numbered long-chain fatty acids levels in a subjectby at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 80%, 85%, 90%, 95%, or 100%, or in a range of from 10% to 50%,20% to 50%, 20% to 75%, 25% to 75%, 25% to 90% or 10% to 99% relative tothe respective concentration prior to administration of a polynucleotide(e.g., mRNA) encoding an MCM polypeptide, as assessed by methods wellknown in the art or described herein. In some embodiments, the methodsfor treating MMA provided herein increase the urinary urea:methylmalonicacid ratio in a subject by at least about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 80%, 85%, 90%, 95%, or 100%, or in arange of from 10% to 50%, 20% to 50%, 20% to 75%, 25% to 75%, 25% to 90%or 10% to 99% relative to the respective concentration prior toadministration of a polynucleotide (e.g., mRNA) encoding an MCMpolypeptide, as assessed by methods well known in the art or describedherein.

In some embodiments, the methods for treating MMA provided hereinincrease the cellular enzyme activity in a subject by at least about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 80%, 85%,90%, 95%, or 100%, or in a range of from 10% to 50%, 20% to 50%, 20% to75%, 25% to 75%, 25% to 90% or 10% to 99% relative to the respectiveconcentration prior to administration of a polynucleotide (e.g., mRNA)encoding an MCM polypeptide, as assessed by methods well known in theart or described herein. In certain embodiment, the increase in cellularenzyme activity is determined by obtaining cells (e.g., fibroblasts orlymphocytes) from the subject, culturing the cells in the presence orabsence of a polynucleotide, and comparing the cellular enzyme activityin the presence of the polynucleotide (e.g., mRNA) encoding an MCMpolypeptide to the cellular enzyme activity in the absence of thepolynucleotide. Techniques for measuring cellular enzyme activity areknown in the art and described herein (see, e.g., Section 6, infra).

In some aspects, the methods for treating MMA provided herein improve ordevelopmental or cognitive function in a subject. In some aspects, themethods for treating MMA provided herein improve control of musclecontractions by a subject as assessed by methods well known in the art.In some embodiments, the methods for treating MMA provided hereinimprove renal function In some embodiments, the methods for treating MMAprovided herein decrease the need for kidney transplant, livertransplant or both. In some embodiments, the methods for treating MMAprovided herein decrease the requirement for hospitalization. In someembodiments, the methods for treating MMA provided herein decrease thelength and/or frequency of hospitalization.

In some embodiments, the subject is a male human. In some embodiments,the subject is a female human. In some embodiments, a subject treatedfor MMA in accordance with the methods provided herein is a fetus. Inaccordance with this embodiment, a pregnant female may be administered apolynucleotide in a manner that permits the polynucleotide to passthrough the placenta to the fetus. Alternatively, the polynucleotide maybe administered directly to the fetus by, e.g., injection. In someembodiments, a subject treated for MMA in accordance with the methodsprovided herein is a human infant. In one embodiment, a subject treatedfor MMA in accordance with the methods provided herein is an elderlyhuman. In another embodiment, a subject treated for MMA in accordancewith the methods provided herein is a human adult. In anotherembodiment, a subject treated for MMA in accordance with the methodsprovided herein is a human child. In another embodiment, a subjecttreated for MMA in accordance with the methods provided herein is ahuman toddler.

In a specific embodiment, a subject treated for MMA in accordance withthe methods provided herein is a human that is less than 5 years old. Inanother specific embodiment, a subject treated for MMA in accordancewith the methods provided herein is a human that is older than 5 yearsold. In a specific embodiment, a subject treated for MMA in accordancewith the methods provided herein is a human that is less than 5 yearsold, is older than 5 years old, is 18 years old or is older than 18years old.

In some embodiments, a subject treated for MMA in accordance with themethods provided herein is administered a polynucleotide (e.g., mRNA)encoding an MCM polypeptide or a pharmaceutical composition thereof, ora combination therapy before any adverse effects or intolerance totherapies other than the polynucleotide develops. In some embodiments, asubject treated for MMA in accordance with the methods provided hereinis a refractory patient. In a certain embodiment, a refractory patientis an MMA patient that is refractory to a standard therapy (e.g.,carnitine or cobalamin supplements).

In some embodiments, a subject treated for MMA in accordance with themethods provided herein is a human that has proven refractory totherapies other than treatment with a polynucleotide, but is no longeron these therapies. In some embodiments, a subject treated for MMA inaccordance with the methods provided herein is a human already receivingone or more conventional MMA therapies, such as camitine supplements,cobalamin supplements, antibiotics, kidney transplant, and/or livertransplant. In some embodiments, a subject treated for MMA in accordancewith the methods provided herein is a human on a low-protein diet. Insome embodiments, a subject treated for MMA in accordance with themethods provided herein is a human on a diet that avoids substancescontaining isoleucine, threonine, methionine, and valine.

In some embodiments, a subject treated for MMA in accordance with themethods provided herein is a human susceptible to adverse reactions toconventional therapies. In some embodiments, a subject treated for MMAin accordance with the methods provided herein is a human that has notreceived a therapy, e.g., a carnitine supplement, a cobalaminsupplement, an antibiotic, a kidney transplant, and/or a livertransplant, prior to the administration of a polynucleotide or apharmaceutical composition thereof. In some embodiments, a subjecttreated for MMA in accordance with the methods provided herein is ahuman that has received a therapy prior to administration of apolynucleotide or a pharmaceutical composition thereof. In someembodiments, a subject treated for MMA in accordance with the methodsprovided herein is a human that has experienced adverse side effects tothe prior therapy or the prior therapy was discontinued due tounacceptable levels of toxicity to the human.

In some embodiments, a subject treated for MMA in accordance with themethods provided herein is a human diagnosed with a complete (mut⁰) orpartial (mut⁻) defect in methylmalonyl-CoA mutase (MCM). The geneencoding MCM is referred to as the MUT gene and is located on chromosome6p21.1.

Dosage and Administration

In accordance with the methods for treating MMA provided herein, apolynucleotide (e.g., mRNA) encoding an MCM polypeptide or apharmaceutical composition thereof can be administered to a subject inneed thereof by a variety of routes in amounts which result in abeneficial or therapeutic effect. A polynucleotide (e.g., mRNA) encodingan MCM polypeptide or pharmaceutical composition thereof may beintravenously administered to a subject in need thereof in accordancewith the methods for treating MMA provided herein.

In accordance with the methods for treating MMA provided herein thatinvolve administration of a polynucleotide (e.g., mRNA) encoding an MCMpolypeptide in combination with one or more additional therapies, thepolynucleotide (e.g., mRNA) encoding an MCM polypeptide and one or moreadditional therapies may be administered by the same route or adifferent route of administration.

The dosage and frequency of administration of a polynucleotide (e.g.,mRNA) encoding an MCM polypeptide or a pharmaceutical compositionthereof is administered to a subject in need thereof in accordance withthe methods for treating MMA provided herein will be efficacious whileminimizing any side effects. The exact dosage and frequency ofadministration of a polynucleotide (e.g., mRNA) encoding an MCMpolypeptide or a pharmaceutical composition thereof can be determined bya practitioner, in light of factors related to the subject that requirestreatment. Factors which may be taken into account include the severityof the disease state, general health of the subject, age, weight, andgender of the subject, diet, time and frequency of administration, drugcombination(s), reaction sensitivities, and tolerance/response totherapy. The dosage and frequency of administration of a polynucleotide(e.g., mRNA) encoding an MCM polypeptide or a pharmaceutical compositionthereof may be adjusted over time to provide sufficient levels of thepolynucleotide or to maintain the desired effect.

In some embodiments, a polynucleotide (e.g., mRNA) encoding an MCMpolypeptide or a pharmaceutical composition thereof is administered to asubject in need thereof in accordance with the methods for treating MMAprovided herein at a dosage and a frequency of administration thatachieves one or more of the following: (i) the reduction or ameliorationof the severity of one or more MMA symptoms; (ii) the reduction in theduration of one or more symptoms associated with MMA; (iii) theprevention in the recurrence of a symptom associated with MMA; (iv) thereduction in hospitalization of a subject; (v) a reduction inhospitalization length; (vi) the increase in the survival of a subject;(vii) the enhancement or improvement of the therapeutic effect ofanother therapy; (viii) an improvement in developmental or cognitiveability; (ix) a decrease in the frequency and/or number of metabolicdecompensation episodes; (x) an improvement in control of musclecontraction; (xi) a reduction in mortality; (xii) an increase in thesurvival rate of patients; (xiii) a decrease in hospitalization rate;(xiv) the prevention of the development or onset of one or more symptomsassociated with MMA; (xv) the reduction in the number of symptomsassociated with MMA; (xvi) an decrease in the concentration ofmethylmalonic acid in biological fluids (e.g., plasma or urine); (xvii)a decrease in the concentration of metabolites of methylmalonic acid,such as propionylcarnitine or methylcitrate, in biological fluids (e.g.,plasma or urine); (xviii) a decrease in erythrocyte odd-numberedlong-chain fatty acid levels; (xix) an increase in the urinaryurea:methylmalonic acid ratio; (xx) an increase in symptom-free survivalof MMA patients; (xxi) an improvement in renal function; and (xxii)improvement in quality of life as assessed by methods well known in theart.

In some embodiments, a polynucleotide (e.g., mRNA) encoding an MCMpolypeptide or a pharmaceutical composition thereof is administered to asubject in need thereof in accordance with the methods for treating MMAprovided herein once in a day. In some embodiments, a polynucleotide(e.g., mRNA) encoding an MCM polypeptide or a pharmaceutical compositionthereof is administered to a subject in need thereof in accordance withthe methods for treating MMA provided herein once every two days. Insome embodiments, a polynucleotide (e.g., mRNA) encoding an MCMpolypeptide or a pharmaceutical composition thereof is administered to asubject in need thereof in accordance with the methods for treating MMAprovided herein once every three days. In some embodiments, apolynucleotide (e.g., mRNA) encoding an MCM polypeptide or apharmaceutical composition thereof is administered to a subject in needthereof in accordance with the methods for treating MMA provided hereinonce every four days. In some embodiments, a polynucleotide (e.g., mRNA)encoding an MCM polypeptide or a pharmaceutical composition thereof isadministered to a subject in need thereof in accordance with the methodsfor treating MMA provided herein once every five days. In someembodiments, a polynucleotide (e.g., mRNA) encoding an MCM polypeptideor a pharmaceutical composition thereof is administered to a subject inneed thereof in accordance with the methods for treating MMA providedherein once every six days. In some embodiments, a polynucleotide (e.g.,mRNA) encoding an MCM polypeptide or a pharmaceutical compositionthereof is administered to a subject in need thereof in accordance withthe methods for treating MMA provided herein once every week. In someembodiments, a polynucleotide (e.g., mRNA) encoding an MCM polypeptideor a pharmaceutical composition thereof is administered to a subject inneed thereof in accordance with the methods for treating MMA providedherein once every two weeks. In some embodiments, a polynucleotide(e.g., mRNA) encoding an MCM polypeptide or a pharmaceutical compositionthereof is administered to a subject in need thereof in accordance withthe methods for treating MMA provided herein once every three weeks. Insome embodiments, a polynucleotide (e.g., mRNA) encoding an MCMpolypeptide or a pharmaceutical composition thereof is administered to asubject in need thereof in accordance with the methods for treating MMAprovided herein once every month. In some embodiments, a polynucleotide(e.g., mRNA) encoding an MCM polypeptide or a pharmaceutical compositionthereof is administered to a subject in need thereof in accordance withthe methods for treating MMA provided herein on a monthly schedulesufficient to maintain decreased symptoms in a MMA patient sufferingtherefrom. In some embodiments, provide herein are methods forcontinuous therapy wherein a polynucleotide (e.g., mRNA) encoding an MCMpolypeptide or a pharmaceutical composition thereof is administered to asubject in need thereof in accordance with the methods for treating MMAprovided herein daily for a certain period of time. In some embodiments,a polynucleotide (e.g., mRNA) encoding an MCM polypeptide or apharmaceutical composition thereof is administered continuously insubsequent 24 hour periods daily, weekly, monthly or yearly.

Treatment periods for a course of therapy can span one week, two weeks,three weeks, four weeks, five weeks, six weeks, seven weeks, eightweeks, nine weeks, ten weeks, eleven weeks, twelve weeks, thirteenweeks, fourteen weeks, four months, five months, six months, sevenmonths, eight months, nine months, ten months, eleven months, one year,two years, three years, four years, five years or longer. The treatmentperiods can be interrupted by periods of rest which can span a day, oneweek, two weeks, three weeks, four weeks, five weeks, six weeks, sevenweeks, eight weeks, nine weeks, ten weeks, eleven weeks, twelve weeks,thirteen weeks, fourteen weeks, four months, five months, six months,seven months, eight months, nine months, ten months, eleven months, oneyear, two years, three years, four years, five years or longer. Suchdeterminations can be made by one skilled in the art (e.g., aphysician). In some embodiments, treatment is intermittent, with periodsof treatment being followed by periods of no treatment. Continuoustreatment can be interrupted by one or more days, months, weeks oryears. Continuous treatment can also be followed by a rest periodlasting one or more days, months, weeks or years, with continuoustreatment then resuming after the rest period.

In a particular embodiment, a polynucleotide or a pharmaceuticalcomposition thereof is administered in a dose of about 0.01-10 mg/kg. Ina particular embodiment, a polynucleotide or a pharmaceuticalcomposition thereof is administered in a dose of about 0.01 mg/kg, 0.02mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg, 0.08,mg/kg, 0.09 mg/kg, 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8mg/kg, 0.9 mg/kg, 1.0 mg/kg, about 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg,about 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, about 1.7 mg/kg, 1.8 mg/kg, 1.9mg/kg, about 2.0 mg/kg, 2.1 mg/kg, 2.2 mg/kg, about 2.3 mg/kg, 2.4mg/kg, 2.5 mg/kg, 3.0 mg/kg, 3.5 mg/kg, 4.0 mg/kg or about 5.0 mg/kg. Ina particular embodiment, a polynucleotide or a pharmaceuticalcomposition thereof is administered in a dose of about 0.01-2 mg/kg. Ina particular embodiment, a polynucleotide or a pharmaceuticalcomposition thereof is administered in a dose of about 0.02-1 mg/kg. Ina particular embodiment, a polynucleotide or a pharmaceuticalcomposition thereof is administered in a dose of about 0.02-1 mg/kg. Ina particular embodiment, a polynucleotide or a pharmaceuticalcomposition thereof is administered in a dose of about 0.02-0.5 mg/kg.In a particular embodiment, a polynucleotide or a pharmaceuticalcomposition thereof is administered in a dose of about 0.03-0.5 mg/kg.In a particular embodiment, a polynucleotide or a pharmaceuticalcomposition thereof is administered in a dose of about 0.04-0.5 mg/kg.In a particular embodiment, a polynucleotide or a pharmaceuticalcomposition thereof is administered in a dose of about 0.05-0.5 mg/kg.

In a particular embodiment, a polynucleotide or a pharmaceuticalcomposition thereof is administered in a dose of about 0.02-0.5 mg/kg.In a particular embodiment, a polynucleotide or a pharmaceuticalcomposition thereof is administered in a dose of about 0.02-0.1 or 0.2mg/kg. In a particular embodiment, a polynucleotide or a pharmaceuticalcomposition thereof is administered in a dose of about 0.02-0.1 or 0.2mg/kg. In a particular embodiment, a polynucleotide or a pharmaceuticalcomposition thereof is administered in a dose of about 0.03-0.1 or 0.2mg/kg. In a particular embodiment, a polynucleotide or a pharmaceuticalcomposition thereof is administered in a dose of about 0.04-0.1 or 0.2mg/kg. In a particular embodiment, a polynucleotide or a pharmaceuticalcomposition thereof is administered in a dose of about 0.05-0.1 or 0.2mg/kg.

In other embodiments, an effective dose for the polynucleotide of thedisclosure is 0.1 mg/kg to 1.0 mg/kg, 0.1 mg/kg to 10 mg/kg, 0.1 mg/kgto 2 mg/kg, 0.1 mg/kg to 5 mg/kg, 1 mg/kg to 5 mg/kg, or 1 mg/kg to 3mg/kg. In some embodiments, the effective dose is sufficient to reducethe plasma MMA level after the administration at least 70%, at least75%, at least 80%, at least 85%, at least 90%, or at least 95% comparedto the plasma MMA level prior to the administration. In otherembodiments, the plasma MMA level is reduced about 75% to 85% comparedto the plasma MMA level prior to the administration.

In other embodiments, the effective dose is sufficient to maintain theplasma MMA level after the administration lower than about 5 μmol/L,about 4.5 μmol/L, about 4 μmol/L, about 3.5 μmol/L, about 3 μmol/L,about 2.5 μmol/L, about 2 μmol/L, about 1.5 μmol/L, about 1 μmol/L,about 0.9 μmol/L, about 0.8 μmol/L, about 0.7 μmol/L, about 0.6 μmol/L,about 0.5 μmol/L, about 0.4 μmol/L, about 0.3 μmol/L, or 0.27 μmol/L.

In certain embodiments, the effective dose is sufficient to maintain theurinary MMA level less than 2000 mmol/mol creatinine, less than 1900mmol/mol creatinine, less than 1800 mmol/mol creatinine, less than 1700mmol/mol creatinine, less than 1600 mmol/mol creatinine, less than 1500mmol/mol creatinine, less than 1400 mmol/mol creatinine, less than 1300mmol/mol creatinine, less than 1200 mmol/mol creatinine, less than 1100mmol/mol creatinine, less than 1000 mmol/mol creatinine, 900 mmol/molcreatinine, 800 mmol/mol creatinine, 700 mmol/mol creatinine, 600mmol/mol creatinine, 500 mmol/mol creatinine, 400 mmol/mol creatinine,300 mmol/mol creatinine, 200 mmol/mol creatinine, 100 mmol/molcreatinine, 90 mmol/mol creatinine, 80 mmol/mol creatinine, 70 mmol/molcreatinine, 60 mmol/mol creatinine, 50 mmol/mol creatinine, 40 mmol/molcreatinine, 30 mmol/mol creatinine, 20 mmol/mol creatinine, 10 mmol/molcreatinine, 9 mmol/mol creatinine, 8 mmol/mol creatinine, 7 mmol/molcreatinine, 6 mmol/mol creatinine, 5 mmol/mol creatinine, 4 mmol/molcreatinine, 3 mmol/mol creatinine, 2 mmol/mol creatinine, or 1 mmol/molcreatinine.

In some embodiments, a method for treating MMA presented herein involvesthe administration to a subject in need thereof of one or more doses ofan effective amount of a polynucleotide (e.g., mRNA) encoding an MCMpolypeptide or a pharmaceutical composition, wherein the effectiveamount may or may not be the same for each dose. In some embodiments, afirst dose of a polynucleotide or pharmaceutical composition thereof isadministered to a subject in need thereof for a first period of time,and subsequently, a second dose of a polynucleotide is administered tothe subject for a second period of time. The first dose may be more thanthe second dose, or the first dose may be less than the second dose. Athird dose of a polynucleotide also may be administered to a subject inneed thereof for a third period of time.

The length of time that a subject in need thereof is administered apolynucleotide or a pharmaceutical composition thereof in accordancewith the methods for treating MMA presented herein will be the timeperiod that is determined to be efficacious. In some embodiments, amethod for treating MMA presented herein involves the administration ofa polynucleotide or a pharmaceutical composition thereof for a period oftime until the severity and/or number of symptoms associated with MMAdecrease.

It will be understood that the amounts of a polynucleotide or apharmaceutical composition thereof administered to a patient in needthereof are or can be calculated based upon the actual weight of thepatient in question or the average weight of the patient population inquestion.

Combination Therapy

Presented herein are combination therapies for the treatment of MMAwhich involve the administration of a polynucleotide (e.g., mRNA)encoding an MCM polypeptide in combination with one or more additionaltherapies to a subject in need thereof. In a specific embodiment,presented herein are combination therapies for the treatment of MMAwhich involve the administration of an effective amount of apolynucleotide (e.g., mRNA) encoding an MCM polypeptide in combinationwith an effective amount of another therapy to a subject in needthereof. Specific examples of such other therapies include, but are notlimited to, carnitine supplements (such as L-carnitine), cobalaminsupplements and antibiotics (such as metronidazole).

The combination therapies provided herein involve administrating to asubject to in need thereof a polynucleotide or a pharmaceuticalcomposition thereof in combination with conventional, or known,therapies for MMA. Current therapies for MMA, include camitinesupplements, cobalamin supplements and antibiotics. Other therapies forMMA or a condition associated therewith are aimed at controlling orrelieving symptoms, e.g., anti-seizure medication. Accordingly, in someembodiments, the combination therapies provided herein involveadministrating to a subject to in need thereof a pain reliever, amedication for epileptic seizures, or other therapy aimed at alleviatingor controlling symptoms associated with MMA or a condition associatedtherewith.

In some embodiments, the methods for treating MMA provided hereincomprise administering a polynucleotide (e.g., mRNA) encoding an MCMpolypeptide as a single agent for a period of time prior toadministering the polynucleotide in combination with an additionaltherapy. In some embodiments, the methods for treating MMA providedherein comprise administering an additional therapy alone for a periodof time prior to administering a polynucleotide (e.g., mRNA) encoding anMCM polypeptide in combination with the additional therapy.

In some embodiments, the administration of a polynucleotide (e.g., mRNA)encoding an MCM polypeptide and one or more additional therapies inaccordance with the methods presented herein have an additive effectrelative the administration of the polynucleotide or said one or moreadditional therapies alone. In some embodiments, the administration of apolynucleotide (e.g., mRNA) encoding an MCM polypeptide and one or moreadditional therapies in accordance with the methods presented hereinhave a synergistic effect relative to the administration of thepolynucleotide or said one or more additional therapies alone.

The combination of a polynucleotide (e.g., mRNA) encoding an MCMpolypeptide and one or more additional therapies can be administered toa subject in the same pharmaceutical composition. Alternatively, apolynucleotide (e.g., mRNA) encoding an MCM polypeptide and one or moreadditional therapies can be administered concurrently to a subject inseparate pharmaceutical compositions. A polynucleotide (e.g., mRNA)encoding an MCM polypeptide and one or more additional therapies can beadministered sequentially to a subject in separate pharmaceuticalcompositions. A polynucleotide (e.g., mRNA) encoding an MCM polypeptideand one or more additional therapies may also be administered to asubject by the same or different routes of administration.

In some embodiments, the combination therapies provided herein involveadministering to a subject in need thereof a polynucleotide (e.g., mRNA)encoding an MCM polypeptide or a pharmaceutical composition thereof incombination with one or more of the following: a camitine supplement(e.g., L-camitine), a cobalamin supplement and an antibiotic. In someembodiments, the combination therapies provided herein involveadministering to a subject in need thereof a polynucleotide (e.g., mRNA)encoding an MCM polypeptide or a pharmaceutical composition thereof incombination with an organ transplant (e.g., a kidney, liver or kidneyand liver transplant).

Clinical Objectives

Efficacy of a polynucleotide for treating MMA may be assessed bydetermining the effects of the polynucleotide on reduction of plasmamethylmalonic acid. The efficacy of a polynucleotide for treating MMAmay also be assessed by: (i) determining the effect on urinary levels ofmethylcitrate; (ii) determining the effect on plasma levels ofpropionlycarnitine; (iii) evaluating effects on erythrocyte oddlong-chain fatty acid levels; (iv) determining effects on the urinaryurea:methylmalonic acid ratio; (v) determining the effects on enzymeactivity in cultured fibroblasts and lymphocytes from subjects with MMA,(vi) evaluating the effects on the developmental and cognitive abilityof subjects; (vii) evaluating the effects on the dystonia rating scale;(viii) evaluating the effects on the occurrence of any metabolicdecompensation episodes; (ix) evaluating the safety profile of thepolynucleotide; (x) evaluating compliance with treatment with thepolynucleotide; and (xi) determining the polynucleotide's plasmaexposure over time.

Clinical Endpoints

A primary clinical endpoint for efficacy of a polynucleotide fortreating MMA includes a reduction in plasma methylmalonic acid levels.Other clinical endpoints for the efficacy of a polynucleotide fortreating MMA may include: a reduction in urinary methylmalonic acidlevels;

a reduction in urinary methylcitrate; a reduction in plasmapropionylcarnitine; a reduction in erythrocyte odd long-chain fatty acidlevels; an increase in the urea:methylmalonic acid ratio;

and pharmacokinetic parameters, e.g., time to maximum plasmaconcentration (T_(max)), C_(max), AUC, terminal elimination half-life(t_(1/2)) based on a polynucleotide's plasma concentrations as assessedby a validated bioanalytical method.

Plasma Methylmalonic Acid Levels

Plasma methylmalonic acid levels may be used to indicate theeffectiveness of a polynucleotide to increase the activity of therelevant enzyme or factor (e.g., MCM). Normal plasma methylmalonic acidlevel is about <0.27 μmol/L (Fowler et al., 2008, J. Inherit. Metab.Dis. 31: 350-360). Plasma methylmalonic acid levels are elevated inpatients with MMA, generally in the range of about 100 to about 1.000μmol/L in cobalamin-non responsive patients and about 5 to about 100μmol/L in cobalamin-responsive patients (Venditti, 2007, Gene Reviews).Methylmalonic acid is considered to be nephrotoxic, and central nervoussystem trapping of methylmalonic acid, propionyl-CoA, and 2methylcitrate is considered to be the basis for chronic neurologiccomplications of MMA (Morath et al., 2008, J. Inherit. Metab. Dis. 31:35-43). A decrease by >30% in plasma or urine methylmalonic acid levelshas been considered by some to be a clinically relevant difference(Zwickler et al., 2008, J. Inherit. Metab. Dis. 31: 361-367).

Blood may be collected and plasma methylmalonic acid concentrations maybe determined using a standard gas chromatography/mass spectroscopy(GC/MS) stable-isotope dilution method.

Urinary Methylmalonic Acid Levels

Urinary methylmalonic acid levels may be used to indicate theeffectiveness of a polynucleotide to increase the activity of therelevant enzyme or factor (e.g., MCM). The normal urinary methylmalonicacid level is <4 mmol/mol creatinine (Venditti, 2007, Gene Reviews), andthe level is significantly elevated in MMA. In general, the more severetypes of MMA, mut⁰ and cblB, have higher urinary methylmalonic acidlevels (about 5,000 to >10.000 mmol/mol creatinine) compared to the lesssevere types, mut⁻ and cblA (<1,000 to >5,000 mol/mol creatinine)(Horster et al., 2007, Pediatr. Res. 62: 225-230; Fowler et al., 2008,J. Inherit. Metab. Dis. 31: 350-360). It has been observed that chronicrenal failure does not occur in patients with urinary methylmalonic acidlevels below −2.000 mmol/mol creatinine (Horster et al., 2007, Pediatr.Res. 62: 225-230).

Urine may be collected and urinary methylmalonic acid concentrations maybe determined using a standard gas chromatography/mass spectroscopy(GC/MS) stable-isotope dilution method.

Plasma Propionylcarnitine, Urinary Methylcitrate, Erythrocyte OLCFAs,Urinary Urea:Methylmalonic Acid Ratio

Plasma propionylcamitine and urinary methylcitrate are methylmalonicacid metabolites and may be used to indicate the effectiveness of apolynucleotide (e.g., mRNA) encoding an MCM polypeptide to increase theactivity of the relevant enzyme or factor (e.g., MCM). It has beensuggested that plasma propionylcamitine may be a more useful measurementthan urinary methylmalonic acid in the presence of renal insufficiency(Horster et al., 2007, Pediatr. Res. 62: 225-230). Urinary methylcitratehas been found to be elevated in the setting of elevated methylmalonicacid levels (Fowler et al., 2008, J. Inherit. Metab. Dis. 31: 350-360).Blood and urine may be collected and standard techniques may be used todetermine plasma propionylcamitine levels and urinary methylcitratelevels, respectively.

OLCFAs are measured in erythrocyte membrane lipids and may be used toindicate the effectiveness of a polynucleotide (e.g., mRNA) encoding anMCM polypeptide to increase the activity of the relevant enzyme orfactor (e.g., MCM). Erythrocyte OLCFAs values reflect both the severityof the disease and the quality of the dietary control in MMA (Merineroet al., 2008, J. Inherit. Metab. Dis. 31: 55-66). This parameter isindicative of the propionyl-CoA load of the cells and of long-termmetabolic control in organic acidemias (Merinero et al., 2008, J.Inherit. Metab. Dis. 31: 55-66; Sperl et al., 2000, Eur. J. Pediatr.159: 54-88). A standard method for determination of erythrocyte OLCFAsconcentrations is available.

An increase in urinary urea:methylmalonic acid ratio followingadministration of a polynucleotide (e.g., mRNA) encoding an MCMpolypeptide compared to baseline may indicate an increase in activity ofthe deficient enzyme or factor (MCM, cblA, or cblB). Protein catabolismleads to production of both urea and methylmalonic acid. The values ofthese catabolic products may fluctuate with dietary protein intake. Ifthe source of methylmalonic acid is predominantly natural protein, inpatients with no enzyme activity (e.g., mut⁰ patients) the ratio ofurinary urea:methylmalonic acid is approximately 3.5. If there isresidual enzyme activity (e.g., administration of vitamin B12 tocobalamin-sensitive patients) the ratio is generally >5. Patientsreceiving amino acid supplements will produce more urea thanmethylmalonic acid and will have a urea:MMA ratio>5. However, even inthis category of patients the urea:MMA ratio will increase if theactivity of the deficient enzyme or factor increases, (Valayannopouloset al., Annual Symposium of the Society for the Study of Inborn Errorsof Metabolism, Amsterdam, The Netherlands, September 2004).

VIII. KITS AND DEVICES Kits

The disclosure provides a variety of kits for conveniently and/oreffectively using the claimed nucleotides of the present disclosure.Typically kits will comprise sufficient amounts and/or numbers ofcomponents to allow a user to perform multiple treatments of asubject(s) and/or to perform multiple experiments.

In one aspect, the present disclosure provides kits comprising themolecules (polynucleotides) of the disclosure.

Said kits can be for protein production, comprising a firstpolynucleotides comprising a translatable region. The kit can furthercomprise packaging and instructions and/or a delivery agent to form aformulation composition. The delivery agent can comprise a saline, abuffered solution, a lipidoid or any delivery agent disclosed herein.

In some embodiments, the buffer solution can include sodium chloride,calcium chloride, phosphate and/or EDTA. In another embodiment, thebuffer solution can include, but is not limited to, saline, saline with2 mM calcium, 5% sucrose, 5% sucrose with 2 mM calcium, 5% Mannitol, 5%Mannitol with 2 mM calcium, Ringer's lactate, sodium chloride, sodiumchloride with 2 mM calcium and mannose (See, e.g., U.S. Pub. No.20120258046; herein incorporated by reference in its entirety). In afurther embodiment, the buffer solutions can be precipitated or it canbe lyophilized. The amount of each component can be varied to enableconsistent, reproducible higher concentration saline or simple bufferformulations. The components can also be varied in order to increase thestability of modified RNA in the buffer solution over a period of timeand/or under a variety of conditions. In one aspect, the presentdisclosure provides kits for protein production, comprising: apolynucleotide comprising a translatable region, provided in an amounteffective to produce a desired amount of a protein encoded by thetranslatable region when introduced into a target cell; a secondpolynucleotide comprising an inhibitory nucleic acid, provided in anamount effective to substantially inhibit the innate immune response ofthe cell; and packaging and instructions.

In one aspect, the present disclosure provides kits for proteinproduction, comprising a polynucleotide comprising a translatableregion, wherein the polynucleotide exhibits reduced degradation by acellular nuclease, and packaging and instructions.

In one aspect, the present disclosure provides kits for proteinproduction, comprising a polynucleotide comprising a translatableregion, wherein the polynucleotide exhibits reduced degradation by acellular nuclease, and a mammalian cell suitable for translation of thetranslatable region of the first nucleic acid.

Devices

The present disclosure provides for devices that can incorporatepolynucleotides that encode polypeptides of interest. These devicescontain in a stable formulation the reagents to synthesize apolynucleotide in a formulation available to be immediately delivered toa subject in need thereof, such as a human patient

Devices for administration can be employed to deliver thepolynucleotides of the present disclosure according to single, multi- orsplit-dosing regimens taught herein. Such devices are taught in, forexample, International Application PCT/US2013/30062 filed Mar. 9, 2013,the contents of which are incorporated herein by reference in theirentirety.

Method and devices known in the art for multi-administration to cells,organs and tissues are contemplated for use in conjunction with themethods and compositions disclosed herein as embodiments of the presentdisclosure. These include, for example, those methods and devices havingmultiple needles, hybrid devices employing for example lumens orcatheters as well as devices utilizing heat, electric current orradiation driven mechanisms.

According to the present disclosure, these multi-administration devicescan be utilized to deliver the single, multi- or split dosescontemplated herein. Such devices are taught for example in,International Application PCT/US2013/30062 filed Mar. 9, 2013, thecontents of which are incorporated herein by reference in theirentirety.

In some embodiments, the polynucleotide is administered subcutaneouslyor intramuscularly via at least 3 needles to three different, optionallyadjacent, sites simultaneously, or within a 60 minutes period (e.g.,administration to 4, 5, 6, 7, 8, 9, or 10 sites simultaneously or withina 60 minute period).

Methods and Devices Utilizing Catheters and/or Lumens

Methods and devices using catheters and lumens can be employed toadminister the polynucleotides of the present disclosure on a single,multi- or split dosing schedule. Such methods and devices are describedin International Application PCT/US2013/30062 filed Mar. 9, 2013(Attorney Docket Number M300), the contents of which are incorporatedherein by reference in their entirety.

Methods and Devices Utilizing Electrical Current

Methods and devices utilizing electric current can be employed todeliver the polynucleotides of the present disclosure according to thesingle, multi- or split dosing regimens taught herein. Such methods anddevices are described in International Application PCT/US2013/30062filed Mar. 9, 2013 (Attorney Docket Number M300), the contents of whichare incorporated herein by reference in their entirety.

IX. DEFINITIONS

In order that the present disclosure can be more readily understood,certain terms are first defined. As used in this application, except asotherwise expressly provided herein, each of the following terms shallhave the meaning set forth below. Additional definitions are set forththroughout the application.

The disclosure includes embodiments in which exactly one member of thegroup is present in, employed in, or otherwise relevant to a givenproduct or process. The disclosure includes embodiments in which morethan one, or all of the group members are present in, employed in, orotherwise relevant to a given product or process.

In this specification and the appended claims, the singular forms “a,”“an” and “the” include plural referents unless the context clearlydictates otherwise. The terms “a” (or “an”), as well as the terms “oneor more,” and “at least one” can be used interchangeably herein. Incertain aspects, the term “a” or “an” means “single.” In other aspects,the term “a” or “an” includes “two or more” or “multiple.”

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; Aand C; A and B; B and C; A (alone); B (alone); and C (alone).

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Wherever aspects are described herein with the language “comprising,”otherwise analogous aspects described in terms of “consisting of” and/or“consisting essentially of” are also provided.

Units, prefixes, and symbols are denoted in their Système Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Where a range of values is recited, it is tobe understood that each intervening integer value, and each fractionthereof, between the recited upper and lower limits of that range isalso specifically disclosed, along with each subrange between suchvalues. The upper and lower limits of any range can independently beincluded in or excluded from the range, and each range where either,neither or both limits are included is also encompassed within thedisclosure. Where a value is explicitly recited, it is to be understoodthat values that are about the same quantity or amount as the recitedvalue are also within the scope of the disclosure. Where a combinationis disclosed, each subcombination of the elements of that combination isalso specifically disclosed and is within the scope of the disclosure.Conversely, where different elements or groups of elements areindividually disclosed, combinations thereof are also disclosed. Whereany element of an disclosure is disclosed as having a plurality ofalternatives, examples of that disclosure in which each alternative isexcluded singly or in any combination with the other alternatives arealso hereby disclosed; more than one element of an disclosure can havesuch exclusions, and all combinations of elements having such exclusionsare hereby disclosed.

Nucleotides are referred to by their commonly accepted single-lettercodes. Unless otherwise indicated, nucleic acids are written left toright in 5′ to 3′ orientation. Nucleotides are referred to herein bytheir commonly known one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Accordingly, A represents adenine,C represents cytosine, G represents guanine, T represents thymine, Urepresents uracil.

Amino acids are referred to herein by either their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Unless otherwise indicated, aminoacid sequences are written left to right in amino to carboxyorientation.

At various places in the present specification, substituents ofcompounds of the present disclosure are disclosed in groups or inranges. It is specifically intended that the present disclosure includeeach and every individual subcombination of the members of such groupsand ranges

About: As used herein, the term “about” means +/−10% of the recitedvalue.

Administered in combination: As used herein, the term “administered incombination” or “combined administration” means that two or more agentsare administered to a subject at the same time or within an intervalsuch that there can be an overlap of an effect of each agent on thepatient. In some embodiments, they are administered within about 60, 30,15, 10, 5, or 1 minute of one another. In some embodiments, theadministrations of the agents are spaced sufficiently closely togethersuch that a combinatorial (e.g., a synergistic) effect is achieved.

Amino acid substitution: The term “amino acid substitution” refers toreplacing an amino acid residue present in a parent sequence (e.g., aconsensus sequence) with another amino acid residue. An amino acid canbe substituted in a parent sequence, for example, via chemical peptidesynthesis or through recombinant methods known in the art. Accordingly,a reference to a “substitution at position X” refers to the substitutionof an amino acid present at position X with an alternative amino acidresidue. In some aspects, substitution patterns can be describedaccording to the schema AnY, wherein A is the single letter codecorresponding to the amino acid naturally present at position n, and Yis the substituting amino acid residue. In other aspects, substitutionpatterns can be described according to the schema An(YZ), wherein A isthe single letter code corresponding to the amino acid residuesubstituting the amino acid naturally present at position X, and Y and Zare alternative substituting amino acid residue, i.e., In the context ofthe present disclosure, substitutions (even when they referred to asamino acid substitution) are conducted at the nucleic acid level, i.e.,substituting an amino acid residue with an alternative amino acidresidue is conducted by substituting the codon encoding the first aminoacid with a codon encoding the second amino acid.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans at anystage of development. In some embodiments, “animal” refers to non-humananimals at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In someembodiments, animals include, but are not limited to, mammals, birds,reptiles, amphibians, fish, and worms. In some embodiments, the animalis a transgenic animal, genetically-engineered animal, or a clone.

Approximately: As used herein, the term “approximately,” as applied toone or more values of interest, refers to a value that is similar to astated reference value. In certain embodiments, the term “approximately”refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%,16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%,or less in either direction (greater than or less than) of the statedreference value unless otherwise stated or otherwise evident from thecontext (except where such number would exceed 100% of a possiblevalue).

Associated with: As used herein with respect to a disease, the term“associated with” means that the symptom, measurement, characteristic,or status in question is linked to the diagnosis, development, presence,or progression of that disease. As association may, but need not, becausatively linked to the disease.

When used with respect to two or more moieties, the terms “associatedwith,” “conjugated,” “linked,” “attached,” and “tethered,” when usedwith respect to two or more moieties, means that the moieties arephysically associated or connected with one another, either directly orvia one or more additional moieties that serves as a linking agent, toform a structure that is sufficiently stable so that the moieties remainphysically associated under the conditions in which the structure isused, e.g., physiological conditions. An “association” need not bestrictly through direct covalent chemical bonding. It can also suggestionic or hydrogen bonding or a hybridization based connectivitysufficiently stable such that the “associated” entities remainphysically associated.

Bifunctional: As used herein, the term “bifunctional” refers to anysubstance, molecule or moiety that is capable of or maintains at leasttwo functions. The functions can affect the same outcome or a differentoutcome. The structure that produces the function can be the same ordifferent. For example, bifunctional modified RNAs of the presentdisclosure can encode a cytotoxic peptide (a first function) while thosenucleosides that comprise the encoding RNA are, in and of themselves,cytotoxic (second function). In this example, delivery of thebifunctional modified RNA to a cancer cell would produce not only apeptide or protein molecule that can ameliorate or treat the cancer butwould also deliver a cytotoxic payload of nucleosides to the cell shoulddegradation, instead of translation of the modified RNA, occur.

Biocompatible: As used herein, the term “biocompatible” means compatiblewith living cells, tissues, organs or systems posing little to no riskof injury, toxicity or rejection by the immune system.

Biodegradable: As used herein, the term “biodegradable” means capable ofbeing broken down into innocuous products by the action of livingthings.

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any substance that has activity in abiological system and/or organism. For instance, a substance that, whenadministered to an organism, has a biological effect on that organism,is considered to be biologically active. In particular embodiments, apolynucleotide of the present disclosure can be considered biologicallyactive if even a portion of the polynucleotide is biologically active ormimics an activity considered biologically relevant.

Chimera: As used herein, “chimera” is an entity having two or moreincongruous or heterogeneous parts or regions.

Conservative amino acid substitution: A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art,including basic side chains (e.g., lysine, arginine, or histidine),acidic side chains (e.g., aspartic acid or glutamic acid), unchargedpolar side chains (e.g., glycine, asparagine, glutamine, serine,threonine, tyrosine, or cysteine), nonpolar side chains (e.g., alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine, ortryptophan), beta-branched side chains (e.g., threonine, valine,isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, or histidine). Thus, if an amino acid in a polypeptide isreplaced with another amino acid from the same side chain family, theamino acid substitution is considered to be conservative. In anotheraspect, a string of amino acids can be conservatively replaced with astructurally similar string that differs in order and/or composition ofside chain family members.

Non-conservative amino acid substitutions include those in which (i) aresidue having an electropositive side chain (e.g., Arg, His or Lys) issubstituted for, or by, an electronegative residue (e.g., Glu or Asp),(ii) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by,a hydrophobic residue (e.g., Ala, Leu, Ile, Phe or Val), (iii) acysteine or proline is substituted for, or by, any other residue, or(iv) a residue having a bulky hydrophobic or aromatic side chain (e.g.,Val, His, Ile or Trp) is substituted for, or by, one having a smallerside chain (e.g., Ala or Ser) or no side chain (e.g., Gly).

Other amino acid substitutions can be readily identified by workers ofordinary skill. For example, for the amino acid alanine, a substitutioncan be taken from any one of D-alanine, glycine, beta-alanine,L-cysteine and D-cysteine. For lysine, a replacement can be any one ofD-lysine, arginine, D-arginine, homo-arginine, methionine, D-methionine,ornithine, or D-ornithine. Generally, substitutions in functionallyimportant regions that can be expected to induce changes in theproperties of isolated polypeptides are those in which (i) a polarresidue, e.g., serine or threonine, is substituted for (or by) ahydrophobic residue, e.g., leucine, isoleucine, phenylalanine, oralanine; (ii) a cysteine residue is substituted for (or by) any otherresidue; (iii) a residue having an electropositive side chain, e.g.,lysine, arginine or histidine, is substituted for (or by) a residuehaving an electronegative side chain, e.g., glutamic acid or asparticacid; or (iv) a residue having a bulky side chain, e.g., phenylalanine,is substituted for (or by) one not having such a side chain, e.g.,glycine. The likelihood that one of the foregoing non-conservativesubstitutions can alter functional properties of the protein is alsocorrelated to the position of the substitution with respect tofunctionally important regions of the protein: some non-conservativesubstitutions can accordingly have little or no effect on biologicalproperties.

Conserved: As used herein, the term “conserved” refers to nucleotides oramino acid residues of a polynucleotide sequence or polypeptidesequence, respectively, that are those that occur unaltered in the sameposition of two or more sequences being compared. Nucleotides or aminoacids that are relatively conserved are those that are conserved amongstmore related sequences than nucleotides or amino acids appearingelsewhere in the sequences.

In some embodiments, two or more sequences are said to be “completelyconserved” if they are 100% identical to one another. In someembodiments, two or more sequences are said to be “highly conserved” ifthey are at least 70% identical, at least 80% identical, at least 90%identical, or at least 95% identical to one another. In someembodiments, two or more sequences are said to be “highly conserved” ifthey are about 70% identical, about 80% identical, about 90% identical,about 95%, about 98%, or about 99% identical to one another. In someembodiments, two or more sequences are said to be “conserved” if theyare at least 30% identical, at least 40% identical, at least 50%identical, at least 60% identical, at least 70% identical, at least 80%identical, at least 90% identical, or at least 95% identical to oneanother. In some embodiments, two or more sequences are said to be“conserved” if they are about 30% identical, about 40% identical, about50% identical, about 60% identical, about 70% identical, about 80%identical, about 90% identical, about 95% identical, about 98%identical, or about 99% identical to one another. Conservation ofsequence can apply to the entire length of an polynucleotide orpolypeptide or can apply to a portion, region or feature thereof.

Controlled Release: As used herein, the term “controlled release” refersto a pharmaceutical composition or compound release profile thatconforms to a particular pattern of release to effect a therapeuticoutcome.

Cyclic or Cyclized: As used herein, the term “cyclic” refers to thepresence of a continuous loop. Cyclic molecules need not be circular,only joined to form an unbroken chain of subunits. Cyclic molecules suchas the engineered RNA or mRNA of the present disclosure can be singleunits or multimers or comprise one or more components of a complex orhigher order structure.

Cytotoxic: As used herein, “cytotoxic” refers to killing or causinginjurious, toxic, or deadly effect on a cell (e.g., a mammalian cell(e.g., a human cell)), bacterium, virus, fungus, protozoan, parasite,prion, or a combination thereof.

Delivery: As used herein, “delivery” refers to the act or manner ofdelivering a compound, substance, entity, moiety, cargo or payload.

Delivery Agent: As used herein, “delivery agent” refers to any substancethat facilitates, at least in part, the in vivo delivery of apolynucleotide to targeted cells.

Destabilized: As used herein, the term “destable,” “destabilize,” or“destabilizing region” means a region or molecule that is less stablethan a starting, wild-type or native form of the same region ormolecule.

Detectable label: As used herein, “detectable label” refers to one ormore markers, signals, or moieties that are attached, incorporated orassociated with another entity that is readily detected by methods knownin the art including radiography, fluorescence, chemiluminescence,enzymatic activity, absorbance and the like. Detectable labels includeradioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions,ligands such as biotin, avidin, streptavidin and haptens, quantum dots,and the like. Detectable labels can be located at any position in thepeptides or proteins disclosed herein. They can be within the aminoacids, the peptides, or proteins, or located at the N- or C-termini.

Diastereomer: As used herein, the term “diastereomer,” meansstereoisomers that are not mirror images of one another and arenon-superimposable on one another.

Digest: As used herein, the term “digest” means to break apart intosmaller pieces or components. When referring to polypeptides orproteins, digestion results in the production of peptides.

Distal: As used herein, the term “distal” means situated away from thecenter or away from a point or region of interest.

Dosing regimen: As used herein, a “dosing regimen” is a schedule ofadministration or physician determined regimen of treatment,prophylaxis, or palliative care.

Effective Amount: As used herein, the term “effective amount” of anagent is that amount sufficient to effect beneficial or desired results,for example, clinical results, and, as such, an “effective amount”depends upon the context in which it is being applied. The term“effective amount” can be used interchangeably with “effective dose,”“therapeutically effective amount,” or “therapeutically effective dose.”

Enantiomer: As used herein, the term “enantiomer” means each individualoptically active form of a compound of the disclosure, having an opticalpurity or enantiomeric excess (as determined by methods standard in theart) of at least 80% (i.e., at least 90% of one enantiomer and at most10% of the other enantiomer), at least 90%, or at least 98%.

Encapsulate: As used herein, the term “encapsulate” means to enclose,surround or encase.

Encoded protein cleavage signal: As used herein, “encoded proteincleavage signal” refers to the nucleotide sequence that encodes aprotein cleavage signal.

Engineered: As used herein, embodiments of the disclosure are“engineered” when they are designed to have a feature or property,whether structural or chemical, that varies from a starting point, wildtype or native molecule.

Effective Amount: As used herein, the term “effective amount” of anagent is that amount sufficient to effect beneficial or desired results,for example, clinical results, and, as such, an “effective amount”depends upon the context in which it is being applied. For example, inthe context of administering an agent that treats high cholesterol, aneffective amount of an agent is, for example, an amount sufficient toachieve treatment, as defined herein, of high cholesterol, as comparedto the response obtained without administration of the agent.

Exosome: As used herein, “exosome” is a vesicle secreted by mammaliancells or a complex involved in RNA degradation.

Expression: As used herein, “expression” of a nucleic acid sequencerefers to one or more of the following events: (1) production of an RNAtemplate from a DNA sequence (e.g., by transcription); (2) processing ofan RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or3′ end processing); (3) translation of an RNA into a polypeptide orprotein; and (4) post-translational modification of a polypeptide orprotein.

Feature: As used herein, a “feature” refers to a characteristic, aproperty, or a distinctive element.

Formulation: As used herein, a “formulation” includes at least apolynucleotide and a delivery agent.

Fragment: A “fragment,” as used herein, refers to a portion. Forexample, fragments of proteins can comprise polypeptides obtained bydigesting full-length protein isolated from cultured cells.

Functional: As used herein, a “functional” biological molecule is abiological molecule in a form in which it exhibits a property and/oractivity by which it is characterized.

Homology: As used herein, the term “homology” refers to the overallrelatedness between polymeric molecules, e.g., between nucleic acidmolecules (e.g., DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. In some embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99% identical or similar. The term “homologous” necessarilyrefers to a comparison between at least two sequences (polynucleotide orpolypeptide sequences). In accordance with the disclosure, twopolynucleotide sequences are considered to be homologous if thepolypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%,95%, or even 99% for at least one stretch of at least about 20 aminoacids. In some embodiments, homologous polynucleotide sequences arecharacterized by the ability to encode a stretch of at least 4-5uniquely specified amino acids. For polynucleotide sequences less than60 nucleotides in length, homology is determined by the ability toencode a stretch of at least 4-5 uniquely specified amino acids. Inaccordance with the disclosure, two protein sequences are considered tobe homologous if the proteins are at least about 50%, 60%, 70%, 80%, or90% identical for at least one stretch of at least about 20 amino acids.

Identity: As used herein, the term “identity” refers to the overallrelatedness between polymeric molecules, e.g., between polynucleotidemolecules (e.g., DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Percent identity can be calculated between twoDNA molecules, between two RNA molecules, and between a DNA molecule andan RNA molecule. When DNA and RNA are compared, T is considered to be U(or vice versa).

Calculation of the percent identity of two polynucleotide sequences, forexample, can be performed by aligning the two sequences for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second nucleic acid sequences for optimal alignment andnon-identical sequences can be disregarded for comparison purposes). Incertain embodiments, the length of a sequence aligned for comparisonpurposes is at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, or 100% of thelength of the reference sequence. The nucleotides at correspondingnucleotide positions are then compared. When a position in the firstsequence is occupied by the same nucleotide as the correspondingposition in the second sequence, then the molecules are identical atthat position. The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences,taking into account the number of gaps, and the length of each gap,which needs to be introduced for optimal alignment of the two sequences.The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two nucleotidesequences can be determined using methods such as those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991;each of which is incorporated herein by reference. For example, thepercent identity between two nucleotide sequences can be determinedusing the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), whichhas been incorporated into the ALIGN program (version 2.0) using aPAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4. The percent identity between two nucleotide sequences can,alternatively, be determined using the GAP program in the GCG softwarepackage using an NWSgapdna.CMP matrix. Methods commonly employed todetermine percent identity between sequences include, but are notlimited to those disclosed in Carillo, H., and Lipman, D., SIAM JApplied Math., 48:1073 (1988); incorporated herein by reference.Techniques for determining identity are codified in publicly availablecomputer programs. Exemplary computer software to determine homologybetween two sequences include, but are not limited to, GCG programpackage, Devereux, J., et al., Nucleic Acids Research, 12(1), 387(1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec.Biol., 215, 403 (1990)).

Intact: As used herein, in the context of a polypeptide, the term“intact” means retaining an amino acid corresponding to the wild-typeprotein, e.g., not mutating or substituting the wild-type amino acid.

Isomer: As used herein, the term “isomer” means any tautomer,stereoisomer, enantiomer, or diastereomer of any compound of thedisclosure. It is recognized that the compounds of the disclosure canhave one or more chiral centers and/or double bonds and, therefore,exist as stereoisomers, such as double-bond isomers (i.e., geometric E/Zisomers) or diastereomers (e.g., enantiomers (i.e., (+) or (−)) orcis/trans isomers). According to the disclosure, the chemical structuresdepicted herein, and therefore the compounds of the disclosure,encompass all of the corresponding stereoisomers, that is, both thestereomerically pure form (e.g., geometrically pure, enantiomericallypure, or diastereomerically pure) and enantiomeric and stereoisomericmixtures, e.g., racemates. Enantiomeric and stereoisomeric mixtures ofcompounds of the disclosure can typically be resolved into theircomponent enantiomers or stereoisomers by well-known methods, such aschiral-phase gas chromatography, chiral-phase high performance liquidchromatography, crystallizing the compound as a chiral salt complex, orcrystallizing the compound in a chiral solvent. Enantiomers andstereoisomers can also be obtained from stereomerically orenantiomerically pure intermediates, reagents, and catalysts bywell-known asymmetric synthetic methods.

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, in a Petri dish, etc., rather than within anorganism (e.g., animal, plant, or microbe).

In vivo: As used herein, the term “in vivo” refers to events that occurwithin an organism (e.g., animal, plant, or microbe or cell or tissuethereof).

Isolated: As used herein, the term “isolated” refers to a substance orentity that has been separated from at least some of the components withwhich it was associated (whether in nature or in an experimentalsetting). Isolated substances can have varying levels of purity inreference to the substances from which they have been associated.Isolated substances and/or entities can be separated from at least about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, or more of the other components with which theywere initially associated. In some embodiments, isolated agents are morethan about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, ormore than about 99% pure. As used herein, a substance is “pure” if it issubstantially free of other components. Substantially isolated: By“substantially isolated” is meant that the compound is substantiallyseparated from the environment in which it was formed or detected.Partial separation can include, for example, a composition enriched inthe compound of the present disclosure. Substantial separation caninclude compositions containing at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 95%, at least about 97%, or at least about 99% by weight of thecompound of the present disclosure, or salt thereof.

A polynucleotide, vector, polypeptide, cell, or any compositiondisclosed herein that is “isolated” is a polynucleotide, vector,polypeptide, cell, or composition that is in a form not found in nature.Isolated polynucleotides, vectors, polypeptides, or compositions includethose that have been purified to a degree that they are no longer in aform in which they are found in nature. In some aspects, apolynucleotide, vector, polypeptide, or composition that is isolated issubstantially pure.

Linker: As used herein, a “linker” refers to a group of atoms, e.g.,10-1,000 atoms, and can be comprised of the atoms or groups such as, butnot limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide,sulfonyl, carbonyl, and imine. The linker can be attached to a modifiednucleoside or nucleotide on the nucleobase or sugar moiety at a firstend, and to a payload, e.g., a detectable or therapeutic agent, at asecond end. The linker can be of sufficient length as to not interferewith incorporation into a nucleic acid sequence. The linker can be usedfor any useful purpose, such as to form polynucleotide multimers (e.g.,through linkage of two or more chimeric polynucleotides molecules or IVTpolynucleotides) or polynucleotides conjugates, as well as to administera payload, as described herein. Examples of chemical groups that can beincorporated into the linker include, but are not limited to, alkyl,alkenyl, alkynyl, amido, amino, ether, thioether, ester, alkylene,heteroalkylene, aryl, or heterocyclyl, each of which can be optionallysubstituted, as described herein. Examples of linkers include, but arenot limited to, unsaturated alkanes, polyethylene glycols (e.g.,ethylene or propylene glycol monomeric units, e.g., diethylene glycol,dipropylene glycol, triethylene glycol, tripropylene glycol,tetraethylene glycol, or tetraethylene glycol), and dextran polymers andderivatives thereof. Other examples include, but are not limited to,cleavable moieties within the linker, such as, for example, a disulfidebond (—S—S—) or an azo bond (—N═N—), which can be cleaved using areducing agent or photolysis. Non-limiting examples of a selectivelycleavable bond include an amido bond can be cleaved for example by theuse of tris(2-carboxyethyl)phosphine (TCEP), or other reducing agents,and/or photolysis, as well as an ester bond can be cleaved for exampleby acidic or basic hydrolysis.

MCM Associated Disease: As used herein, an “MCM-associated disease” or“MCM-associated disorder” refers to diseases or disorders, respectively,which results from aberrant MCM activity (e.g., decreased activity orincreased activity). As a non-limiting example, methylmalonic acidemiaan MCM-associated disease.

Mitochondrial transit peptide: As used herein, the terms “mitochondrialtransit peptide,” “mitochondrial targeting peptide,” and “mitochondrialtargeting sequence” refer to an amino acid sequence (or a polynucleotideencoding such an amino acid sequence) that is a part of a largerpolypeptide, where that sequence directs the transport or localizationof the larger polypeptide to mitochondria.

Modified: As used herein “modified” refers to a changed state orstructure of a molecule of the disclosure. Molecules can be modified inmany ways including chemically, structurally, and functionally. In someembodiments, the mRNA molecules of the present disclosure are modifiedby the introduction of non-natural nucleosides and/or nucleotides, e.g.,as it relates to the natural ribonucleotides A, U, G, and C.Noncanonical nucleotides such as the cap structures are not considered“modified” although they differ from the chemical structure of the A, C,G, U ribonucleotides.

Mucus: As used herein, “mucus” refers to the natural substance that isviscous and comprises mucin glycoproteins.

Naturally occurring: As used herein, “naturally occurring” meansexisting in nature without artificial aid.

Non-human vertebrate: As used herein, a “non-human vertebrate” includesall vertebrates except Homo sapiens, including wild and domesticatedspecies. Examples of non-human vertebrates include, but are not limitedto, mammals, such as alpaca, banteng, bison, camel, cat, cattle, deer,dog, donkey, gayal, goat, guinea pig, horse, llama, mule, pig, rabbit,reindeer, sheep water buffalo, and yak.

Nucleic acid sequence: The terms “nucleic acid sequence” and “nucleotidesequence” are used interchangeably and refer to a contiguous nucleicacid sequence. The sequence can be either single stranded or doublestranded DNA or RNA, e.g., an mRNA.

The term “nucleic acid,” in its broadest sense, includes any compoundand/or substance that comprises a polymer of nucleotides. These polymersare often referred to as polynucleotides. Exemplary nucleic acids orpolynucleotides of the disclosure include, but are not limited to,ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleicacids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs),locked nucleic acids (LNAs, including LNA having a β-D-riboconfiguration, α-LNA having an α-L-ribo configuration (a diastereomer ofLNA), 2′-amino-LNA having a 2′-amino functionalization, and2′-amino-α-LNA having a 2′-amino functionalization), ethylene nucleicacids (ENA), cyclohexenyl nucleic acids (CeNA) or hybrids orcombinations thereof.

The phrase “nucleotide sequence encoding” and variants thereof refers tothe nucleic acid (e.g., an mRNA or DNA molecule) coding sequence thatcomprises a nucleotide sequence that encodes a polypeptide or functionalfragment thereof as set forth herein. The coding sequence can furtherinclude initiation and termination signals operably linked to regulatoryelements including a promoter and polyadenylation signal capable ofdirecting expression in the cells of an individual or mammal to whichthe nucleic acid is administered. The coding sequence can furtherinclude sequences that encode signal peptides or targeting peptides,e.g., mitochondrial transit peptides.

Off-target: As used herein, “off target” refers to any unintended effecton any one or more target, gene, or cellular transcript.

Open reading frame: As used herein, “open reading frame” or “ORF” refersto a sequence that does not contain a stop codon in a given readingframe.

Operably linked: As used herein, the phrase “operably linked” refers toa functional connection between two or more molecules, constructs,transcripts, entities, moieties or the like.

Optionally substituted: Herein a phrase of the form “optionallysubstituted X” (e.g., optionally substituted alkyl) is intended to beequivalent to “X, wherein X is optionally substituted” (e.g., “alkyl,wherein said alkyl is optionally substituted”). It is not intended tomean that the feature “X” (e.g., alkyl) per se is optional.

Part: As used herein, a “part” or “region” of a polynucleotide isdefined as any portion of the polynucleotide that is less than theentire length of the polynucleotide.

Peptide: As used herein, “peptide” is less than or equal to 50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long.

Patient: As used herein, “patient” refers to a subject who can seek orbe in need of treatment, requires treatment, is receiving treatment,will receive treatment, or a subject who is under care by a trainedprofessional for a particular disease or condition.

Pharmaceutically acceptable: The phrase “pharmaceutically acceptable” isemployed herein to refer to those compounds, materials, compositions,and/or dosage forms that are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem or complication, commensurate with a reasonablebenefit/risk ratio.

Pharmaceutically acceptable excipients: The phrase “pharmaceuticallyacceptable excipient,” as used herein, refers any ingredient other thanthe compounds described herein (for example, a vehicle capable ofsuspending or dissolving the active compound) and having the propertiesof being substantially nontoxic and non-inflammatory in a patient.Excipients can include, for example: antiadherents, antioxidants,binders, coatings, compression aids, disintegrants, dyes (colors),emollients, emulsifiers, fillers (diluents), film formers or coatings,flavors, fragrances, glidants (flow enhancers), lubricants,preservatives, printing inks, sorbents, suspensing or dispersing agents,sweeteners, and waters of hydration. Exemplary excipients include, butare not limited to: butylated hydroxytoluene (BHT), calcium carbonate,calcium phosphate (dibasic), calcium stearate, croscarmellose,crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol,methionine, methylcellulose, methyl paraben, microcrystalline cellulose,polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinizedstarch, propyl paraben, retinyl palmitate, shellac, silicon dioxide,sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate,sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide,vitamin A, vitamin E, vitamin C, and xylitol.

Pharmaceutically acceptable salts: The present disclosure also includespharmaceutically acceptable salts of the compounds described herein. Asused herein, “pharmaceutically acceptable salts” refers to derivativesof the disclosed compounds wherein the parent compound is modified byconverting an existing acid or base moiety to its salt form (e.g., byreacting the free base group with a suitable organic acid). Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. Representative acid addition salts include acetate, acetic acid,adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzenesulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate,camphorsulfonate, citrate, cyclopentanepropionate, digluconate,dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate,glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like, aswell as nontoxic ammonium, quaternary ammonium, and amine cations,including, but not limited to ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like. The pharmaceutically acceptablesalts of the present disclosure include the conventional non-toxic saltsof the parent compound formed, for example, from non-toxic inorganic ororganic acids. The pharmaceutically acceptable salts of the presentdisclosure can be synthesized from the parent compound that contains abasic or acidic moiety by conventional chemical methods. Generally, suchsalts can be prepared by reacting the free acid or base forms of thesecompounds with a stoichiometric amount of the appropriate base or acidin water or in an organic solvent, or in a mixture of the two;generally, nonaqueous media like ether, ethyl acetate, ethanol,isopropanol, or acetonitrile are used. Lists of suitable salts are foundin Remington's Pharmaceutical Sciences, 17^(th) ed., Mack PublishingCompany, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties,Selection, and Use, P. H. Stahl and C. G. Wermuth (eds.), Wiley-VCH,2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19(1977), each of which is incorporated herein by reference in itsentirety.

Pharmaceutically acceptable solvate: The term “pharmaceuticallyacceptable solvate,” as used herein, means a compound of the disclosurewherein molecules of a suitable solvent are incorporated in the crystallattice. A suitable solvent is physiologically tolerable at the dosageadministered. For example, solvates can be prepared by crystallization,recrystallization, or precipitation from a solution that includesorganic solvents, water, or a mixture thereof. Examples of suitablesolvents are ethanol, water (for example, mono-, di-, and tri-hydrates),N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO),N,N′-dimethylformamide (DMF), N,N′-dimethylacetamide (DMAC),1,3-dimethyl-2-imidazolidinone (DMEU),1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile(ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone,benzyl benzoate, and the like. When water is the solvent, the solvate isreferred to as a “hydrate.”

Pharmacokinetic: As used herein, “pharmacokinetic” refers to any one ormore properties of a molecule or compound as it relates to thedetermination of the fate of substances administered to a livingorganism. Pharmacokinetics is divided into several areas including theextent and rate of absorption, distribution, metabolism and excretion.This is commonly referred to as ADME where: (A) Absorption is theprocess of a substance entering the blood circulation; (D) Distributionis the dispersion or dissemination of substances throughout the fluidsand tissues of the body; (M) Metabolism (or Biotransformation) is theirreversible transformation of parent compounds into daughtermetabolites; and (E) Excretion (or Elimination) refers to theelimination of the substances from the body. In rare cases, some drugsirreversibly accumulate in body tissue.

Physicochemical: As used herein, “physicochemical” means of or relatingto a physical and/or chemical property.

Polynucleotide: The term “polynucleotide” as used herein refers topolymers of nucleotides of any length, including ribonucleotides,deoxyribonucleotides, analogs thereof, or mixtures thereof. This termrefers to the primary structure of the molecule. Thus, the term includestriple-, double- and single-stranded deoxyribonucleic acid (“DNA”), aswell as triple-, double- and single-stranded ribonucleic acid (“RNA”).It also includes modified, for example by alkylation, and/or by capping,and unmodified forms of the polynucleotide. More particularly, the term“polynucleotide” includes polydeoxyribonucleotides (containing2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), includingtRNA, rRNA, hRNA, siRNA and mRNA, whether spliced or unspliced, anyother type of polynucleotide that is an N- or C-glycoside of a purine orpyrimidine base, and other polymers containing normucleotidic backbones,for example, polyamide (e.g., peptide nucleic acids “PNAs”) andpolymorpholino polymers, and other synthetic sequence-specific nucleicacid polymers providing that the polymers contain nucleobases in aconfiguration that allows for base pairing and base stacking, such as isfound in DNA and RNA. In particular aspects, the polynucleotidecomprises an mRNA. In other aspect, the mRNA is a synthetic mRNA. Insome aspects, the synthetic mRNA comprises at least one unnaturalnucleobase. In some aspects, all nucleobases of a certain class havebeen replaced with unnatural nucleobases (e.g., all uridines in apolynucleotide disclosed herein can be replaced with an unnaturalnucleobase, e.g., 5-methoxyuridine). In some aspects, the polynucleotide(e.g., a synthetic RNA or a synthetic DNA) comprises only naturalnucleobases, i.e., A, C, T and U in the case of a synthetic DNA, or A,C, T, and U in the case of a synthetic RNA.

The skilled artisan will appreciate that the T bases in the codon mapsdisclosed herein are present in DNA, whereas the T bases would bereplaced by U bases in corresponding RNAs. For example, acodon-nucleotide sequence disclosed herein in DNA form, e.g., a vectoror an in-vitro translation (IVT) template, would have its T basestranscribed as U based in its corresponding transcribed mRNA. In thisrespect, both sequence-optimized DNA sequences (comprising T) and theircorresponding RNA sequences (comprising U) are consideredsequence-optimized nucleotide sequence of the present disclosure. Askilled artisan would also understand that equivalent codon-maps can begenerated by replaced one or more bases with non-natural bases. Thus,e.g., a TTC codon (DNA map) would correspond to a UUC codon (RNA map),which in turn would correspond to a ΨΨC codon (RNA map in which U hasbeen replaced with pseudouridine).

Standard A-T and G-C base pairs form under conditions that allow theformation of hydrogen bonds between the N3-H and C4-oxy of thymidine andthe N1 and C6-NH2, respectively, of adenosine and between the C2-oxy, N3and C4-NH2, of cytidine and the C2-NH2, N′—H and C6-oxy, respectively,of guanosine. Thus, for example, guanosine(2-amino-6-oxy-9-β-D-ribofuranosyl-purine) can be modified to formisoguanosine (2-oxy-6-amino-9-β-D-ribofuranosyl-purine). Suchmodification results in a nucleoside base that will no longereffectively form a standard base pair with cytosine. However,modification of cytosine (1-β-D-ribofuranosyl-2-oxy-4-amino-pyrimidine)to form isocytosine (1-β-D-ribofuranosyl-2-amino-4-oxy-pyrimidine-)results in a modified nucleotide that will not effectively base pairwith guanosine but will form a base pair with isoguanosine (U.S. Pat.No. 5,681,702 to Collins et al., hereby incorporated by reference in itsentirety). Isocytosine is available from Sigma Chemical Co. (St. Louis,Mo.); isocytidine can be prepared by the method described by Switzer etal. (1993) Biochemistry 32:10489-10496 and references cited therein;2′-deoxy-5-methyl-isocytidine can be prepared by the method of Tor etal., 1993, J. Am. Chem. Soc. 115:4461-4467 and references cited therein;and isoguanine nucleotides can be prepared using the method described bySwitzer et al., 1993, supra, and Mantsch et al., 1993, Biochem.14:5593-5601, or by the method described in U.S. Pat. No. 5,780,610 toCollins et al., each of which is hereby incorporated by reference in itsentirety. Other nonnatural base pairs can be synthesized by the methoddescribed in Piccirilli et al., 1990, Nature 343:33-37, herebyincorporated by reference in its entirety, for the synthesis of2,6-diaminopyrimidine and its complement(1-methylpyrazolo-[4,3]pyrimidine-5,7-(4H,6H)-dione. Other such modifiednucleotide units that form unique base pairs are known, such as thosedescribed in Leach et al. (1992) J. Am. Chem. Soc. 114:3675-3683 andSwitzer et al., supra.

Polypeptide: As used herein, “polypeptide” means a polymer of amino acidresidues (natural or unnatural) linked together most often by peptidebonds. The term, as used herein, refers to proteins, polypeptides, andpeptides of any size, structure, or function. Thus, polypeptides includegene products, naturally occurring polypeptides, synthetic polypeptides,homologs, orthologs, paralogs, fragments and other equivalents,variants, and analogs of the foregoing. A polypeptide can be a singlemolecule or can be a multi-molecular complex such as a dimer, trimer ortetramer. They can also comprise single chain or multichain polypeptidesand can be associated or linked. Most commonly disulfide linkages arefound in multichain polypeptides. The term polypeptide can also apply toamino acid polymers in which one or more amino acid residues are anartificial chemical analogue of a corresponding naturally occurringamino acid.

The term polypeptide encompasses an amino acid polymer that has beenmodified naturally or by intervention; for example, disulfide bondformation, glycosylation, lipidation, acetylation, phosphorylation, orany other manipulation or modification, such as conjugation with alabeling component. Also included within the definition are, forexample, polypeptides containing one or more analogs of an amino acid(including, for example, unnatural amino acids such as homocysteine,ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as wellas other modifications known in the art.

Polypeptide variant: As used herein, the term “polypeptide variant”refers to molecules that differ in their amino acid sequence from anative or reference sequence. The amino acid sequence variants canpossess substitutions, deletions, and/or insertions at certain positionswithin the amino acid sequence, as compared to a native or referencesequence. Ordinarily, variants will possess at least about 50% identity(homology), at least about 60% identity, at least about 70% identity, atleast about 80% identity, at least about 90% identity, at least about95% identity, at least about 99% identity to a native or referencesequence. In some embodiments, they will be at least about 80%, or atleast about 90% identical (homologous) to a native or referencesequence.

Polypeptide per unit drug (PUD): As used herein, a PUD or product perunit drug, is defined as a subdivided portion of total daily dose,usually 1 mg, pg, kg, etc., of a product (such as a polypeptide) asmeasured in body fluid or tissue, usually defined in concentration suchas pmol/mL, mmol/mL, etc. divided by the measure in the body fluid.

Preventing: As used herein, the term “preventing” refers to partially orcompletely delaying onset of an infection, disease, disorder and/orcondition; partially or completely delaying onset of one or moresymptoms, features, or clinical manifestations of a particularinfection, disease, disorder, and/or condition; partially or completelydelaying onset of one or more symptoms, features, or manifestations of aparticular infection, disease, disorder, and/or condition; partially orcompletely delaying progression from an infection, a particular disease,disorder and/or condition; and/or decreasing the risk of developingpathology associated with the infection, the disease, disorder, and/orcondition.

Prodrug: The present disclosure also includes prodrugs of the compoundsdescribed herein. As used herein, “prodrugs” refer to any substance,molecule or entity that is in a form predicate for that substance,molecule or entity to act as a therapeutic upon chemical or physicalalteration. Prodrugs can by covalently bonded or sequestered in some wayand that release or are converted into the active drug moiety prior to,upon or after administered to a mammalian subject. Prodrugs can beprepared by modifying functional groups present in the compounds in sucha way that the modifications are cleaved, either in routine manipulationor in vivo, to the parent compounds. Prodrugs include compounds whereinhydroxyl, amino, sulfhydryl, or carboxyl groups are bonded to any groupthat, when administered to a mammalian subject, cleaves to form a freehydroxyl, amino, sulfhydryl, or carboxyl group respectively. Preparationand use of prodrugs is discussed in T. Higuchi and V. Stella, “Pro-drugsas Novel Delivery Systems,” Vol. 14 of the A.C.S. Symposium Series, andin Bioreversible Carriers in Drug Design, ed. Edward B. Roche, AmericanPharmaceutical Association and Pergamon Press, 1987, both of which arehereby incorporated by reference in their entirety.

Proliferate: As used herein, the term “proliferate” means to grow,expand or increase or cause to grow, expand or increase rapidly.“Proliferative” means having the ability to proliferate.“Anti-proliferative” means having properties counter to or inapposite toproliferative properties.

Prophylactic: As used herein, “prophylactic” refers to a therapeutic orcourse of action used to prevent the spread of disease.

Prophylaxis: As used herein, a “prophylaxis” refers to a measure takento maintain health and prevent the spread of disease. An “immunephrophylaxis” refers to a measure to produce active or passive immunityto prevent the spread of disease.

Protein cleavage site: As used herein, “protein cleavage site” refers toa site where controlled cleavage of the amino acid chain can beaccomplished by chemical, enzymatic or photochemical means.

Protein cleavage signal: As used herein “protein cleavage signal” refersto at least one amino acid that flags or marks a polypeptide forcleavage.

Protein of interest: As used herein, the terms “proteins of interest” or“desired proteins” include those provided herein and fragments, mutants,variants, and alterations thereof.

Proximal: As used herein, the term “proximal” means situated nearer tothe center or to a point or region of interest.

Pseudouridine: As used herein, pseudouridine refers to the C-glycosideisomer of the nucleoside uridine. A “pseudouridine analog” is anymodification, variant, isoform or derivative of pseudouridine. Forexample, pseudouridine analogs include but are not limited to1-carboxymethyl-pseudouridine, 1-propynyl-pseudouridine,1-taurinomethyl-pseudouridine, 1-taurinomethyl-4-thio-pseudouridine,1-methylpseudouridine (m¹ψ), 1-methyl-4-thio-pseudouridine (m¹s⁴ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydropseudouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine,N1-methyl-pseudouridine,1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ ψ), and2′-O-methyl-pseudouridine (ψm).

Purified: As used herein, “purify,” “purified,” “purification” means tomake substantially pure or clear from unwanted components, materialdefilement, admixture or imperfection.

Repeated transfection: As used herein, the term “repeated transfection”refers to transfection of the same cell culture with a polynucleotide aplurality of times. The cell culture can be transfected at least twice,at least 3 times, at least 4 times, at least 5 times, at least 6 times,at least 7 times, at least 8 times, at least 9 times, at least 10 times,at least 11 times, at least 12 times, at least 13 times, at least 14times, at least 15 times, at least 16 times, at least 17 times at least18 times, at least 19 times, at least 20 times, at least 25 times, atleast 30 times, at least 35 times, at least 40 times, at least 45 times,at least 50 times or more.

Sample: As used herein, the term “sample” or “biological sample” refersto a subset of its tissues, cells or component parts (e.g., body fluids,including but not limited to blood, mucus, lymphatic fluid, synovialfluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood,urine, vaginal fluid and semen). A sample further can include ahomogenate, lysate or extract prepared from a whole organism or a subsetof its tissues, cells or component parts, or a fraction or portionthereof, including but not limited to, for example, plasma, serum,spinal fluid, lymph fluid, the external sections of the skin,respiratory, intestinal, and genitourinary tracts, tears, saliva, milk,blood cells, tumors, organs. A sample further refers to a medium, suchas a nutrient broth or gel, which can contain cellular components, suchas proteins or nucleic acid molecule.

Signal Sequences: As used herein, the phrase “signal sequences” refersto a sequence that can direct the transport or localization of aprotein.

Single unit dose: As used herein, a “single unit dose” is a dose of anytherapeutic administered in one dose/at one time/single route/singlepoint of contact, i.e., single administration event.

Similarity: As used herein, the term “similarity” refers to the overallrelatedness between polymeric molecules, e.g., between polynucleotidemolecules (e.g., DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of percent similarity of polymericmolecules to one another can be performed in the same manner as acalculation of percent identity, except that calculation of percentsimilarity takes into account conservative substitutions as isunderstood in the art.

Split dose: As used herein, a “split dose” is the division of singleunit dose or total daily dose into two or more doses.

Stable: As used herein “stable” refers to a compound that issufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and in some cases capable of formulation intoan efficacious therapeutic agent.

Stabilized: As used herein, the term “stabilize,” “stabilized,”“stabilized region” means to make or become stable.

Stereoisomer: As used herein, the term “stereoisomer” refers to allpossible different isomeric as well as conformational forms that acompound can possess (e.g., a compound of any formula described herein),in particular all possible stereochemically and conformationallyisomeric forms, all diastereomers, enantiomers and/or conformers of thebasic molecular structure. Some compounds of the present disclosure canexist in different tautomeric forms, all of the latter being includedwithin the scope of the present disclosure.

Subject: As used herein, the term “subject” or “patient” refers to anyorganism to which a composition in accordance with the disclosure can beadministered, e.g., for experimental, diagnostic, prophylactic, and/ortherapeutic purposes. Typical subjects include animals (e.g., mammalssuch as mice, rats, rabbits, non-human primates, and humans).

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Substantially equal: As used herein as it relates to time differencesbetween doses, the term means plus/minus 2%.

Substantially simultaneously: As used herein and as it relates toplurality of doses, the term means within 2 seconds.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with or displays one ormore symptoms of a disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition has not been diagnosed with and/or can notexhibit symptoms of the disease, disorder, and/or condition but harborsa propensity to develop a disease or its symptoms. In some embodiments,an individual who is susceptible to a disease, disorder, and/orcondition (for example, cancer) can be characterized by one or more ofthe following: (1) a genetic mutation associated with development of thedisease, disorder, and/or condition; (2) a genetic polymorphismassociated with development of the disease, disorder, and/or condition;(3) increased and/or decreased expression and/or activity of a proteinand/or nucleic acid associated with the disease, disorder, and/orcondition; (4) habits and/or lifestyles associated with development ofthe disease, disorder, and/or condition; (5) a family history of thedisease, disorder, and/or condition; and (6) exposure to and/orinfection with a microbe associated with development of the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition will develop thedisease, disorder, and/or condition. In some embodiments, an individualwho is susceptible to a disease, disorder, and/or condition will notdevelop the disease, disorder, and/or condition.

Sustained release: As used herein, the term “sustained release” refersto a pharmaceutical composition or compound release profile thatconforms to a release rate over a specific period of time.

Synthetic: The term “synthetic” means produced, prepared, and/ormanufactured by the hand of man. Synthesis of polynucleotides orpolypeptides or other molecules of the present disclosure can bechemical or enzymatic.

Targeted Cells: As used herein, “targeted cells” refers to any one ormore cells of interest. The cells can be found in vitro, in vivo, insitu or in the tissue or organ of an organism. The organism can be ananimal, for example a mammal, a human or a patient.

Targeting sequence: As used herein, the phrase “targeting sequence”refers to a sequence that can direct the transport or localization of aprotein.

Therapeutic Agent: The term “therapeutic agent” refers to any agentthat, when administered to a subject, has a therapeutic, diagnostic,and/or prophylactic effect and/or elicits a desired biological and/orpharmacological effect.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” means an amount of an agent to bedelivered (e.g., nucleic acid, drug, therapeutic agent, diagnosticagent, prophylactic agent, etc.) that is sufficient, when administeredto a subject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve symptoms of, diagnose,prevent, and/or delay the onset of the infection, disease, disorder,and/or condition.

Therapeutically effective outcome: As used herein, the term“therapeutically effective outcome” means an outcome that is sufficientin a subject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve symptoms of, diagnose,prevent, and/or delay the onset of the infection, disease, disorder,and/or condition.

Total daily dose: As used herein, a “total daily dose” is an amountgiven or prescribed in 24 hr. period. It can be administered as a singleunit dose.

Transcription factor: As used herein, the term “transcription factor”refers to a DNA-binding protein that regulates transcription of DNA intoRNA, for example, by activation or repression of transcription. Sometranscription factors effect regulation of transcription alone, whileothers act in concert with other proteins. Some transcription factor canboth activate and repress transcription under certain conditions. Ingeneral, transcription factors bind a specific target sequence orsequences highly similar to a specific consensus sequence in aregulatory region of a target gene. Transcription factors can regulatetranscription of a target gene alone or in a complex with othermolecules.

Transcription: As used herein, the term “transcription” refers tomethods to introduce exogenous nucleic acids into a cell. Methods oftransfection include, but are not limited to, chemical methods, physicaltreatments and cationic lipids or mixtures.

Treating: As used herein, the terms “treating” or “treatment” or“therapy” refers to partially or completely alleviating, ameliorating,improving, relieving, delaying onset of, inhibiting progression of,reducing severity of, and/or reducing incidence of one or more symptomsor features of a particular infection, disease, disorder, and/orcondition. Treatment can be administered to a subject who does notexhibit signs of a disease, disorder, and/or condition and/or to asubject who exhibits only early signs of a disease, disorder, and/orcondition for the purpose of decreasing the risk of developing pathologyassociated with the disease, disorder, and/or condition.

Unmodified: As used herein, “unmodified” refers to any substance,compound or molecule prior to being changed in any way. Unmodified may,but does not always, refer to the wild type or native form of abiomolecule. Molecules can undergo a series of modifications wherebyeach modified molecule can serve as the “unmodified” starting moleculefor a subsequent modification.

Viral protein: As used herein, the phrase “viral protein” means anyprotein originating from a virus.

X. CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/269,089 filed Dec. 17, 2015, entitled Polynucleotides EncodingMethylmalonyl-CoA Mutase; U.S. Provisional Patent Application No.62/269,092 filed Dec. 17, 2015, entitled Methods of Using MCM-EncondingPolynucleotides; U.S. Provisional Patent Application No. 62/273,112filed Dec. 30, 2015, entitled Polynucleotides Encoding Methylmalonyl-CoAMutase; U.S. Provisional Patent Application No. 62/273,108 filed Dec.30, 2015, entitled Methods of Using MCM-Enconding Polynucleotides; U.S.Provisional Patent Application No. 62/274,727 filed Jan. 4, 2016,entitled Polynucleotides Encoding Methylmalonyl-CoA Mutase; U.S.Provisional Patent Application No. 62/274,733 filed Jan. 4, 2016,entitled Methods of Using MCM-Enconding Polynucleotides; U.S.Provisional Patent Application No. 62/274,722 filed Jan. 4, 2016,entitled Polynucleotides Encoding Methylmalonyl-CoA Mutase; U.S.Provisional Patent Application No. 62/274,726 filed Jan. 4, 2016,entitled Methods of Using MCM-Enconding Polynucleotides; U.S.Provisional Patent Application No. 62/338,478 filed Can 18, 2016,entitled Polynucleotides Encoding Methylmalonyl-CoA Mutase; U.S.Provisional Patent Application No. 62/338,456 filed Can 18, 2016,entitled Polynucleotides Encoding Methylmalonyl-CoA Mutase; and U.S.Provisional Patent Application No. 62/409,343 filed Oct. 14, 2016,entitled Polynucleotides Encoding Methylmalonyl-CoA Mutase, the contentsof each of which are herein incorporated by reference in its entirety.

XI. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing (Name:“3529_052PC07_SeqListing_ST25.txt”; Size: 1,080,572 bytes; and date ofcreation: Dec. 16, 2016) filed herewith the application is incorporatedby reference in its entirety.

XII. EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the disclosure described herein. Thescope of the present disclosure is not intended to be limited to theabove Description, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” can mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The disclosure includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Thedisclosure includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

It is also noted that the term “comprising” is intended to be open andpermits but does not require the inclusion of additional elements orsteps. When the term “comprising” is used herein, the term “consistingof” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the disclosure, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present disclosure that falls within the prior art can be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they can beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the disclosure (e.g., anynucleic acid or protein encoded thereby; any method of production; anymethod of use; etc.) can be excluded from any one or more claims, forany reason, whether or not related to the existence of prior art.

All cited sources, for example, references, publications, databases,database entries, and art cited herein, are incorporated into thisapplication by reference, even if not expressly stated in the citation.In case of conflicting statements of a cited source and the instantapplication, the statement in the instant application shall control.

Section and table headings are not intended to be limiting.

EXAMPLES Example 1. Chimeric Polynucleotide Synthesis Triphosphate Route

Two regions or parts of a chimeric polynucleotide can be joined orligated using triphosphate chemistry. According to this method, a firstregion or part of 100 nucleotides or less can be chemically synthesizedwith a 5′ monophosphate and terminal 3′desOH or blocked OH. If theregion is longer than 80 nucleotides, it can be synthesized as twostrands for ligation.

If the first region or part is synthesized as a non-positionallymodified region or part using in vitro transcription (IVT), conversionthe 5′monophosphate with subsequent capping of the 3′ terminus canfollow. Monophosphate protecting groups can be selected from any ofthose known in the art.

The second region or part of the chimeric polynucleotide can besynthesized using either chemical synthesis or IVT methods. IVT methodscan include an RNA polymerase that can utilize a primer with a modifiedcap. Alternatively, a cap of up to 80 nucleotides can be chemicallysynthesized and coupled to the IVT region or part.

It is noted that for ligation methods, ligation with DNA T4 ligase,followed by treatment with DNAse should readily avoid concatenation.

The entire chimeric polynucleotide need not be manufactured with aphosphate-sugar backbone. If one of the regions or parts encodes apolypeptide, then such region or part can comprise a phosphate-sugarbackbone.

Ligation can then be performed using any known click chemistry,orthoclick chemistry, solulink, or other bioconjugate chemistries knownto those in the art.

Synthetic Route

The chimeric polynucleotide can be made using a series of startingsegments. Such segments include:

-   -   (a) Capped and protected 5′ segment comprising a normal 3′OH        (SEG. 1)    -   (b) 5′ triphosphate segment which can include the coding region        of a polypeptide and comprising a normal 3′OH (SEG. 2)    -   (c) 5′ monophosphate segment for the 3′ end of the chimeric        polynucleotide (e.g., the tail) comprising cordycepin or no 3′OH        (SEG. 3)

After synthesis (chemical or IVT), segment 3 (SEG. 3) can be treatedwith cordycepin and then with pyrophosphatase to create the5′monophosphate.

Segment 2 (SEG. 2) can then be ligated to SEG. 3 using RNA ligase. Theligated polynucleotide can then be purified and treated withpyrophosphatase to cleave the diphosphate. The treated SEG.2-SEG. 3construct is then purified and SEG. 1 is ligated to the 5′ terminus. Afurther purification step of the chimeric polynucleotide can beperformed.

Where the chimeric polynucleotide encodes a polypeptide, the ligated orjoined segments can be represented as: 5′UTR (SEG. 1), open readingframe or ORF (SEG. 2) and 3′UTR+PolyA (SEG. 3).

The yields of each step can be as much as 90-95%.

Example 2: PCR for cDNA Production

PCR procedures for the preparation of cDNA can be performed using 2×KAPAHIFI™ HotStart ReadyMix by Kapa Biosystems (Woburn, Mass.). This systemincludes 2×KAPA ReadyMix 12.5 μl; Forward Primer (10 μM) 0.75 μl;Reverse Primer (10 μM) 0.75 μl; Template cDNA −100 ng; and dH₂0 dilutedto 25.0 μl. The PCR reaction conditions can be: at 95° C. for 5 min. and25 cycles of 98° C. for 20 sec, then 58° C. for 15 sec, then 72° C. for45 sec, then 72° C. for 5 min. then 4° C. to termination.

The reverse primer of the instant disclosure can incorporate a poly-T120for a poly-A₁₂₀ in the mRNA. Other reverse primers with longer orshorter poly(T) tracts can be used to adjust the length of the poly(A)tail in the polynucleotide mRNA.

The reaction can be cleaned up using Invitrogen's PURELINK™ PCR MicroKit (Carlsbad, Calif.) per manufacturer's instructions (up to 5 μg).Larger reactions will require a cleanup using a product with a largercapacity. Following the cleanup, the cDNA can be quantified using theNANODROP™ and analyzed by agarose gel electrophoresis to confirm thecDNA is the expected size. The cDNA can then be submitted for sequencinganalysis before proceeding to the in vitro transcription reaction.

Example 3. In Vitro Transcription (IVT)

The in vitro transcription reactions can generate polynucleotidescontaining uniformly modified polynucleotides. Such uniformly modifiedpolynucleotides can comprise a region or part of the polynucleotides ofthe disclosure. The input nucleotide triphosphate (NTP) mix can be madeusing natural and un-natural NTPs.

A typical in vitro transcription reaction can include the following:

-   -   1 Template cDNA—1.0 μg    -   2 10× transcription buffer (400 mM Tris-HCl pH 8.0, 190 mM        MgCl₂, 50 mM DTT, 10 mM Spermidine)—2.0 μl    -   3 Custom NTPs (25 mM each)—7.2 μl    -   4 RNase Inhibitor—20 U    -   5 T7 RNA polymerase—3000 U    -   6 dH₂0—Up to 20.0 μl. and    -   7 Incubation at 37° C. for 3 hr-5 hrs.

The crude IVT mix can be stored at 4° C. overnight for cleanup the nextday. 1 U of RNase-free DNase can then be used to digest the originaltemplate. After 15 minutes of incubation at 37° C., the mRNA can bepurified using Ambion's MEGACLEAR™ Kit (Austin, Tex.) following themanufacturer's instructions. This kit can purify up to 500 μg of RNA.Following the cleanup, the RNA can be quantified using the NanoDrop andanalyzed by agarose gel electrophoresis to confirm the RNA is the propersize and that no degradation of the RNA has occurred.

Example 4. Enzymatic Capping

Capping of a polynucleotide can be performed with a mixture includes:IVT RNA 60 μg-180 μg and dH₂0 up to 72 μl. The mixture can be incubatedat 65° C. for 5 minutes to denature RNA, and then can be transferredimmediately to ice.

The protocol can then involve the mixing of 10× Capping Buffer (0.5 MTris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl₂) (10.0 μl); 20 mM GTP (5.0μl); 20 mM S-Adenosyl Methionine (2.5 μl); RNase Inhibitor (100 U);2′-O-Methyltransferase (400U); Vaccinia capping enzyme (Guanylyltransferase) (40 U); dH₂0 (Up to 28 μl); and incubation at 37° C. for 30minutes for 60 μg RNA or up to 2 hours for 180 μg of RNA.

The polynucleotide can then be purified using Ambion's MEGACLEAR™ Kit(Austin, Tex.) following the manufacturer's instructions. Following thecleanup, the RNA can be quantified using the NANODROP™ (ThermoFisher,Waltham, Mass.) and analyzed by agarose gel electrophoresis to confirmthe RNA is the proper size and that no degradation of the RNA hasoccurred. The RNA product can also be sequenced by running areverse-transcription-PCR to generate the cDNA for sequencing.

Example 5. PolyA Tailing Reaction

Without a poly-T in the cDNA, a poly-A tailing reaction must beperformed before cleaning the final product. This can be done by mixingCapped IVT RNA (100 μl); RNase Inhibitor (20 U); 10× Tailing Buffer (0.5M Tris-HCl (pH 8.0), 2.5 M NaCl, 100 mM MgCl₂)(12.0 μl); 20 mM ATP (6.0μl); Poly-A Polymerase (20 U); dH₂0 up to 123.5 μl and incubating at 37°C. for 30 min. If the poly-A tail is already in the transcript, then thetailing reaction can be skipped and proceed directly to cleanup withAmbion's MEGACLEAR™ kit (Austin, Tex.) (up to 500 μg). Poly-A Polymeraseis, in some cases, a recombinant enzyme expressed in yeast.

It should be understood that the processivity or integrity of the polyAtailing reaction does not always result in an exact size polyA tail.Hence polyA tails of approximately between 40-200 nucleotides, e.g.,about 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 150-165, 155, 156,157, 158, 159, 160, 161, 162, 163, 164 or 165 are within the scope ofthe disclosure.

Example 6. Natural 5′ Caps and 5′ Cap Analogues

5′-capping of polynucleotides can be completed concomitantly during thein vitro-transcription reaction using the following chemical RNA capanalogs to generate the 5′-guanosine cap structure according tomanufacturer protocols: 3′-O-Me-m7G(5′)ppp(5′) G [the ARCAcap];G(5′)ppp(5′)A; G(5′)ppp(5′)G; m7G(5′)ppp(5′)A; m7G(5′)ppp(5′)G (NewEngland BioLabs, Ipswich, Mass.). 5′-capping of modified RNA can becompleted post-transcriptionally using a Vaccinia Virus Capping Enzymeto generate the “Cap 0” structure: m7G(5′)ppp(5′)G (New England BioLabs,Ipswich, Mass.). Cap 1 structure can be generated using both VacciniaVirus Capping Enzyme and a 2′-O methyl-transferase to generate:m7G(5′)ppp(5′)G-2′-O-methyl. Cap 2 structure can be generated from theCap 1 structure followed by the 2′-O-methylation of the5′-antepenultimate nucleotide using a 2′-O methyl-transferase. Cap 3structure can be generated from the Cap 2 structure followed by the2′-O-methylation of the 5′-preantepenultimate nucleotide using a 2′-Omethyl-transferase. Enzymes can be derived from a recombinant source.

When transfected into mammalian cells, the modified mRNAs can have astability of between 12-18 hours or more than 18 hours, e.g., 24, 36,48, 60, 72 or greater than 72 hours.

Example 7. Capping Assays A. Protein Expression Assay

Polynucleotides encoding a polypeptide, containing any of the capstaught herein, can be transfected into cells at equal concentrations.After 6, 12, 24 and 36 hours post-transfection, the amount of proteinsecreted into the culture medium can be assayed by ELISA. Syntheticpolynucleotides that secrete higher levels of protein into the mediumwould correspond to a synthetic polynucleotide with a highertranslationally-competent Cap structure.

B. Purity Analysis Synthesis

Polynucleotides encoding a polypeptide, containing any of the capstaught herein, can be compared for purity using denaturing Agarose-Ureagel electrophoresis or HPLC analysis. Polynucleotides with a single,consolidated band by electrophoresis correspond to the higher purityproduct compared to polynucleotides with multiple bands or streakingbands. Synthetic polynucleotides with a single HPLC peak would alsocorrespond to a higher purity product. The capping reaction with ahigher efficiency would provide a more pure polynucleotide population.

C. Cytokine Analysis

Polynucleotides encoding a polypeptide, containing any of the capstaught herein, can be transfected into cells at multiple concentrations.After 6, 12, 24 and 36 hours post-transfection the amount ofpro-inflammatory cytokines such as TNF-alpha and IFN-beta secreted intothe culture medium can be assayed by ELISA. Polynucleotides resulting inthe secretion of higher levels of pro-inflammatory cytokines into themedium would correspond to polynucleotides containing animmune-activating cap structure.

D. Capping Reaction Efficiency

Polynucleotides encoding a polypeptide, containing any of the capstaught herein, can be analyzed for capping reaction efficiency by LC-MSafter nuclease treatment. Nuclease treatment of capped polynucleotideswould yield a mixture of free nucleotides and the capped5′-5-triphosphate cap structure detectable by LC-MS. The amount ofcapped product on the LC-MS spectra can be expressed as a percent oftotal polynucleotide from the reaction and would correspond to cappingreaction efficiency. The cap structure with higher capping reactionefficiency would have a higher amount of capped product by LC-MS.

Example 8. Agarose Gel Electrophoresis of Modified RNA or RT PCRProducts

Individual polynucleotides (200-400 ng in a 20 μl volume) or reversetranscribed PCR products (200-400 ng) can be loaded into a well on anon-denaturing 1.2% Agarose E-Gel (Invitrogen, Carlsbad, Calif.) and runfor 12-15 minutes according to the manufacturer protocol.

Example 9. Nanodrop Modified RNA Quantification and UV Spectral Data

Modified polynucleotides in TE buffer (1 μl) can be used for Nanodrop UVabsorbance readings to quantitate the yield of each polynucleotide froman chemical synthesis or in vitro transcription reaction.

Example 10. Formulation of Modified mRNA Using Lipidoids

Polynucleotides can be formulated for in vitro experiments by mixing thepolynucleotides with the lipidoid at a set ratio prior to addition tocells. In vivo formulation can require the addition of extra ingredientsto facilitate circulation throughout the body. To test the ability ofthese lipidoids to form particles suitable for in vivo work, a standardformulation process used for siRNA-lipidoid formulations can be used asa starting point. After formation of the particle, polynucleotide can beadded and allowed to integrate with the complex. The encapsulationefficiency can be determined using a standard dye exclusion assays.

Example 11. Method of Screening for Protein Expression A. ElectrosprayIonization

A biological sample that can contain proteins encoded by apolynucleotide administered to the subject can be prepared and analyzedaccording to the manufacturer protocol for electrospray ionization (ESI)using 1, 2, 3 or 4 mass analyzers. A biologic sample can also beanalyzed using a tandem ESI mass spectrometry system.

Patterns of protein fragments, or whole proteins, can be compared toknown controls for a given protein and identity can be determined bycomparison.

B. Matrix-Assisted Laser Desorption/Ionization

A biological sample that can contain proteins encoded by one or morepolynucleotides administered to the subject can be prepared and analyzedaccording to the manufacturer protocol for matrix-assisted laserdesorption/ionization (MALDI).

Patterns of protein fragments, or whole proteins, can be compared toknown controls for a given protein and identity can be determined bycomparison.

C. Liquid Chromatography-Mass Spectrometry-Mass Spectrometry

A biological sample, which can contain proteins encoded by one or morepolynucleotides, can be treated with a trypsin enzyme to digest theproteins contained within. The resulting peptides can be analyzed byliquid chromatography-mass spectrometry-mass spectrometry (LC/MS/MS).The peptides can be fragmented in the mass spectrometer to yielddiagnostic patterns that can be matched to protein sequence databasesvia computer algorithms. The digested sample can be diluted to achieve 1ng or less starting material for a given protein. Biological samplescontaining a simple buffer background (e.g., water or volatile salts)are amenable to direct in-solution digest; more complex backgrounds(e.g., detergent, non-volatile salts, glycerol) require an additionalclean-up step to facilitate the sample analysis.

Patterns of protein fragments, or whole proteins, can be compared toknown controls for a given protein and identity can be determined bycomparison.

Example 12. Synthesis of mRNA Encoding MCM

The sequence optimized polynucleotides encoding MCM polypeptides, i.e.,SEQ ID NOs: 1-207, 732-765, and 772, are synthesized as described inExamples 1 to 12.

Further, mRNA's encoding both mouse MCM and human MCM were prepared forExamples 13-19 described below, and they were synthesized as describedin Examples 1 to 12.

An mRNA encoding human MCM (“hMCM-mRNA”) was constructed that encodesthe naturally-occurring V671 mutation of MCM. The nucleotide sequence ofhMCM-mRNA is provided in SEQ ID NO: 249. The hMCM-mRNA sequence includesboth 5′ and 3′ UTR regions (SEQ ID NOs: 266 and 267, respectively):

5′UTR: TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCA CC 3′UTR:TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC

A mRNA encoding mouse MCM (“mMCM-mRNA”) was also constructed usingstandard molecular biology techniques. The nucleotide sequence ofmMCM-mRNA is provided in SEQ ID NO: 250, and it encodes mouse MCM (SEQID NO: 268). The mMCM-mRNA sequence includes the same 5′ and 3′ UTRregions as hMCM-mRNA.

Both hMCM-mRNA and mMCM-mRNA were prepared as modified mRNA.Specifically, during in vitro translation, modified mRNA was generatedusing 1-methyl-pseudoUTP to ensure that the mRNAs contained 100%1-methyl-pseudouridine instead of uridine. Further, both hMCM-mRNA andmMCM-mRNA were synthesized with a primer that introduced a 100nucleotide polyA-tail, and a Cap 1 structure was generated on both mRNAsusing Vaccinia Virus Capping Enzyme and a 2′-O methyl-transferase togenerate: m7G(5′)ppp(5′)G-2′-O-methyl.

Example 13. Detecting Endogenous MCM Expression In Vitro

MCM expression was characterized in a variety of cell lines, includingcells derived from both mice and human sources. The cell lines testedincluded Hepa1-6 (mouse), HepG2 (human), SNU423 (human), and HeLa cells.Cell were cultured in standard conditions and cell extracts wereobtained by placing the cells in lysis buffer. For comparison purposes,a mitochondrial extract from mouse liver was also prepared. To preparethe liver extract, whole liver was homogenized in an ice-coldsucrose-containing hypotonic buffer at neutral pH adjusted with 0.1molarTrizma base-MOPS (3-(N-morpholino)propanesulfonic acid). The liverhomogenates were centrifuged at 600 g in a cold table-top centrifuge toremove the nuclear fraction. The mitochondrial fraction was thencollected by centrifugation of the cleared lysates at 7000 g.

To analyze MCM expression, 15 μg of each lysate was prepared in a 40 μLvolume with lithium dodecyl sulfate sample loading buffer and subjectedto standard Western blot analysis. For detection of MCM, the antibodyused was anti-methylmalonyl-CoA mutase (mouse polyclonal; Ab67869;ABCAM®) at a 1:1000 dilution. For detection of a load control, theantibody used was anti-citrase synthase (rabbit polyclonal; PA5-22126;Thermo-Fisher SCIENTIFIC®). In FIG. 1, the signal provided by anti-MCMis provided in green, while the anti-citrate synthase signal is providedin red. FIG. 1 shows that MCM was found in all cell lines tested,including both mouse and human liver-derived cell lines. For all celllines, the expression of MCM was in roughly the same proportion to thecontrol citrate synthase signal, though less signal was observed in HeLacells. FIG. 1 also shows that the cell lines expressed MCM at a levelcomparable to mouse liver mitochondrial mitochondrial extract.

To examine the localization of endogenous MCM, immunofluorescenceanalysis was performed on HeLa cells. MCM expression was detected usinganti-methylmalonyl-CoA mutase (mouse monoclonal; Ab67869; ABCAM®),mitochondria were detected using Mitotracker, and the nucleus wasstained with DAPI. Image analysis was performed on a Zeiss ELYRA imagingsystem. As seen in FIGS. 2(A)-(C), the HeLa immunofluorescent stainingreveals extensive colocalization between mitochondria (red) and MCMimmunofluorescence (green), with little to no colocalization between thenucleus (DAPI stain) and MCM. The finding that the majority ofendogenous MCM localizes with mitochondria is consistent with MCM'sknown metabolic function and localization.

Example 14. In Vitro Expression of MCM in HeLa Cells

To measure in vitro expression of human MCM in HeLa cells, those cellswere seeded on 12-well plates (BD Biosciences, San Jose, USA) one dayprior to transfection. mRNA formulations comprising human MCM (SEQ IDNO: 249) or a GFP control (encoding SEQ ID NO: 269) were transfectedusing 800 ng mRNA and 2 μL Lipofectamin 2000 in 60 μL OPTI-MEM per welland incubated. After 24 hours, the cells in each well were lysed using aconsistent amount of lysis buffer. For comparison purposes, mouse livermitochondrial extract was also prepared as described in Example 13.Protein concentrations of each were determined using a BCA assayaccording to manufacturer's instructions. To analyze MCM expression,equal load of each lysate (24 μg) was prepared in a loading buffer andsubjected to standard Western blot analysis. For detection of MCM, theantibody used was anti-methylmalonyl-CoA mutase (rabbit monoclonal;ab133672; Abcam®) at a 1:1000 dilution.

The resulting Western blot, shown in FIG. 3, demonstrates thatintroduction of a mRNA formulation comprising hMCM sequence (SEQ ID NO:249) greatly increased the level of MCM expression in HeLa cellsrelative to cells transfected with a control construct. The MCMexpression level observed after introduction of mRNA encoding MCM wereeven higher than the levels present in liver mitochondrial mitochondrialextract. While FIG. 1 indicates that at least some endogenous MCM ispresent in HeLa cells, FIG. 3 shows that the MCM expression levels farexceed that baseline after introduction of mRNAs comprising hMCM.

Example 15. In Vitro MCM Activity in HeLa Cells

While the Western blots of FIG. 3 demonstrate that MCM is expressedafter introduction of mRNA comprising an MCM sequence, FIG. 3 does notaddress whether the exogenously-expressed MCM is active. To answer thisquestion, an in vitro MCM activity assay was performed.

A. Expression

HeLa cells were transfected with mRNA formulations comprising human MCM(SEQ ID NO: 249) or a GFP control (encoding SEQ ID NO: 269) weretransfected with Lipofectamin 2000 and lysed as described in Example 14above. For comparison purposes, mouse liver mitochondrial extract wasalso prepared as described in Example 13 above.

B. Activity Assay

To assess whether exogenous MCM can function, an in vitro activity assaywas performed using transfected HeLa cell lysates as the source ofenzymatic activity. To begin, 60 μL of lysate containing 132 μg proteinwas mixed with 30 μL of MCM coenzyme adocobalimin (1 mM in distilledwater) at 37° C. for 5 minutes. Equal amounts ofDL-2-[methyl-¹⁴C]-methylmalonyl-CoA (50-60 mCi per mmol; Cat ARC0847;American Radiolabeled Chemicals) were then added to each reaction, whichwas further incubated at 37° C. for 10 minutes. The reaction was stoppedby adding 50 μL of 100 g/L TCA and vortexing. The reaction tubes werethen centrifuged at 13,000 g for 1 min, and the supernatant was analyzedfor the presence of [¹⁴C]-succinyl-CoA using HPLC-based separation andquantification. Specifically, 20 μL of each activity reactionsupernatant was analyzed using a HPLC system equipped with aQuaternary-Pump, a Multi-sampler, a Thermostated Column-Compartment, aPoroshell EC-C18 120 HPLC-column and a Radiometric Detector controlledby OpenLAB Chromatography Data System, all used according to themanufacturers' recommendations. Elusion was with a linear methanolgradient: 0-15 min (Solvent A: 95% 100 mM acetic acid in 100 mM sodiumphosphate buffer, pH 7.0) and 15-25 min (95% Solvent A with 5% of an 18%v/v methanol/water solution) with a flow rate of 0.5 mL/min.

FIG. 4 shows that there was very low to no MCM activity detected incontrol transfected HeLa cells. FIG. 4 also shows, however, thattransfection with mRNA encoding hMCM led to MCM activity of almost 0.2mM/min/mg. The MCM activity of cell lysate generated by transfectionwith mRNA encoding hMCM was roughly half of that observed in mouse livermitochondrial extract.

Example 16. Measuring In Vitro Expression of MCM

Hepa1-6 cells and fibroblasts from normal subject (NHDF) and MMApatients (GM50 and GM1673) were examined for their capacity to expressexogenous MCM.

Cells were transfected with mRNA formulations comprising human MCM (SEQID NO: 249), mouse MCM (SEQ ID NO: 250), or a GFP control (encoding SEQID NO: 269) via electroporation using a standard protocol. Eachconstruct was tested separately. After 24 hours incubation, cells werelysed and protein concentration in each lysate was measured by BCAassay. To analyze MCM expression, equal load of each lysate (26 μg) wasprepared in a loading buffer and subjected to standard Western blotanalysis. For detection of MCM, the antibody used wasanti-methylmalonyl-CoA mutase (mouse monoclonal; Anti-MUT TRUEMABAntibody Clone OTI2C8; OriGene®) at a 1:5000 dilution. For detection ofa load control, the antibody used was anti-citrase synthase (rabbitpolyclonal; MA5-17625; Pierce®).

FIG. 5 shows that introduction of formulations containing a human MCMsequence (SEQ ID NO: 249) greatly increased the level of MCM expressionin normal human cells and in cells from MMA patients, relative to cellstransfected with a control construct. The increased MCM expression levelobserved after introduction of mRNA encoding hMCM were reflected in theappearance in FIG. 5 of an MCM band in cells transfected with mRNAencoding hMCM. While no significant difference in MCM levels was notedafter transfection with mouse MCM (SEQ ID NO: 250), this could be due toa specific interaction of the MCM antibody with only the human form ofthe enzyme.

FIGS. 6A-6D show co-localization of MCM and mitochondria in humanfibroblasts transfected with eGFP or MCM mRNAs. To examine thelocalization of mRNA-encoded hMCM in mitochondria, 1×10⁶ MCM-deficientpatient fibroblasts (GM01673) were transfected with 1 μg of hMCM mRNA.24 hours after transfection, the cells were incubated with 200 nMMitoTracker Red CMXRos (M7512, ThermoFisher Scientific) for 30 min tomark mitochondria and stained with anti-MCM mouse mAb (TA506873,Origene) to examine the cellular localization.). FIGS. 6A and 6C areimages taken of patient fibroblasts transfected with mRNA encoding eGFP.FIGS. 6B and 6D are images taken of patient fibroblasts transfected withmRNA encoding hMCM. A co-localization of MCM and mitochondria wasobserved, suggesting expressed MCM proteins reside inside mitochondria.

Example 17. Measuring In Vitro MCM Activity A. Expression

Hepa1-6 cells and fibroblasts from normal human subject (NHDF) and MMApatients (GM50 and GM1673) were cultured. Cells were transfected withmRNA formulations comprising human MCM (SEQ ID NO: 249), mouse MCM (SEQID NO: 250), or a GFP control (encoding SEQ ID NO: 269) viaelectroporation using a standard protocol.

B. Activity Assay

To assess whether exogenous MCM can function, an in vitro activity assaywas performed using transfected cell lysates as the source of enzymaticactivity. To begin, 60 μL of lysate containing 100 μg protein was mixedwith 30 μL of MCM coenzyme adocobalimin (1 mM in distilled water) at 37°C. for 5 minutes. Equal amounts of DL-2-[methyl-¹⁴C]-methylmalonyl-CoA(50-60 mCi per mmol; Cat ARC0847; American Radiolabeled Chemicals) werethen added to each reaction, which was further incubated at 37° C. for10 minutes. The reaction was stopped by adding 50 μL of 100 g/L TCA andvortexing. The reaction tubes were then centrifuged at 13,000 g for 1min, and the supernatant was analyzed for the presence of[¹⁴C]-succinyl-CoA using HPLC-based separation and quantification.Specifically, 20 μL of each activity reaction supernatant was analyzedusing a HPLC system equipped with a Quaternary-Pump, a Multi-sampler, aThermostated Column-Compartment, a Poroshell EC-C18 120 HPLC-column anda Radiometric Detector controlled by OpenLAB Chromatography Data System,all used according to the manufacturers' recommendations. Elusion waswith a linear methanol gradient: 0-15 min (Solvent A: 95% 100 mM aceticacid in 100 mM sodium phosphate buffer, pH 7.0) and 15-25 min (95%Solvent A with 5% of an 18% v/v methanol/water solution) with a flowrate of 0.5 mL/min.

FIG. 7 shows that, for each cell lines tested, transfection with mRNA'sencoding either mouse MCM or human MCM increased the level of MCMactivity in all cell types tested. Thus this data demonstrates thattransfection of mRNA encoding MCM leads to the expression of active MMenzyme in Hepa1-6 cells, fibroblasts from normal human subjects, andfibroblasts from MMA patients. Further, human MCM increased MCM activityto a greater degree than did mouse MCM when transfected into Hepa1-6cells.

Example 18. Increased In Vivo MCM Expression

To assess the ability of MCM-containing mRNA's to facilitate MCMexpression in vivo, mRNA encoding human MCM (SEQ ID NO: 249) wasintroduced into C57B/L6 mice. C57B/L6 mice were injected intravenouslywith either control mRNA (NT-FIX) or hMCM mRNA at 0.5 mg/kg. The mRNAwas formulated in lipid nanoparticles for delivery into the mice. Micewere sacrificed after 24 or 48 hrs. and MCM protein levels in liverlysates were determined by capillary electrophoresis (CE). Citratesynthase expression was examined for use as a loading control. Forcontrol NT-FIX injections, 4 mice were tested for each time point. ForhMCM mRNA injections, 6 mice were tested for each time point.

As shown in FIGS. 8A and 8B, MCM expression was drastically increased inall mice injected with mRNA encoding human MCM. The MCM expressionpeaked at the 24 hour time point, and was still higher than control miceat 48 hours. Relatively low levels of variance were observed betweenmice of each experimental condition, indicating that treatment with mRNAencoding MCM is capable of reliably inducing expression of MCM.

Example 19. Human MCM Mutant Constructs

According to the present disclosure, the polynucleotide can comprise atleast a first region of linked nucleosides encoding human MCM. Exemplaryhuman MCM protein sequences of the present disclosure are listed inTable 3 above.

Example 20. MMA Levels and Body Weight Change in MCK Mouse Model

A MCK mouse model of MMA (Mut−/−;Tg^(INS-MCK-Mut)) is known in theliterature and lacks exon 3 of the MCM allele, which encodes thesubstrate-binding pocket of MCM. Manoli 2013 (PNAS 110(33):13552-13557(2013); and Harrington et al., “Stable Isotope Breath Tests to AssessMetabolite Flux in Methylmalonic Acidemia (MMA)”, American Society ofHuman Genetics, Abstract, 2014, which are incorporated herein byreference in their entireties. The MCM knockout mice exhibit asemipenetrant neonatal lethal phenotype, with most mice perishing in theearly neonatal period. This mouse model is described in WO 2014/143884A2, hereby incorporated by reference in its entirety. Because the severephenotype associated with the knockout makes study difficult,researchers have prepared partial rescues of the MCM knockout mice bytransgenic rescue with tissue-specific expression or expression ofmutants that mimic those mutations associated with human MMA. Forexample, transgenic mice that express MCM in the knockout backgroundunder the control of the muscle-specific creatine kinase promoter (“theMCK mice”) survive but still display severe metabolic perturbations andgrowth abnormalities.

The effectiveness of mRNA encoding human MCM was assessed in the MCKmouse model of MMA (Mut−/−;Tg^(INS-MCK-Mut)), N=3-4. MCK mice wereinjected intravenously with either control mRNA (NT-FIX) or codonoptimized human MCM mRNA at 0.16 mg/kg. Five (5) injections were donewith MCM mRNA (SEQ ID NO: 734, FIG. 9) at 0.16 mg/kg; and two injectionswere done with MCM mRNA (SEQ ID NO: 735, FIG. 10) at 0.2 mg/kg. The mRNAwas formulated in lipid nanoparticles (Compound 18) for delivery intothe mice via tail vein injection. Plasma was collected twice a week, 3and 6 days after dosing. Plasma MMA was determined by LC-MS/MS. Bodyweight was measured twice a week at time of dosing. Mice were notsacrificed at the end of this study and were used for other studies. Onthe third scheduled injection (day 15), one of the mice that hadpreviously been administered control mRNA was switched to beingadministered hMCM mRNA and the other mouse administered control mRNA wasfound dead. This testing demonstrated that repeat intravenous dosing ofhMCM mRNA corrected biochemical and growth abnormalities in an animalmodel of Methylmalonic acidemia (MMA-emia).

Although expression of MCM in skeletal muscle rescues MCK mice fromneonatal lethality, these mice display severe metabolic perturbationsand growth retardation that resembles clinical characteristics observedin methylmalonic acidemia patients. In the present example, repeat IVdosing of Compound 18 LNP-encapsulated hMCM mRNA (fully modified withmo5U) was investigated to determine whether mRNA therapy can correctbiochemical and growth abnormalities. MCK mice were administered weeklyIV injections of 0.16 mg/kg hMCM mRNA (encoding SEQ ID NO: 734) or avehicle control (non-translating factor IX, NTFIX) mRNA. MCK mice thatreceived hMCM mRNA injections showed decreased plasma MMA levels 3 and 6days following each dose throughout the entire treatment period (FIG.16A). Importantly, treated MCK mice also showed increased weight gainwhich was correlated to decreased plasma MMA levels (FIG. 16B). Incontrast, MCK mice that received vehicle mRNA control (NTFIX) showedneither reduction in plasma MMA levels nor increase in body weightfollowing 2 doses (FIG. 16A). Indeed, after 2 IV doses of NTFIXinjections, one of the MCK mice died likely due to metabolicdecompensation although the cause of death is unknown. To presentfurther loss of MCK mouse, the vehicle control (NTFIX) mRNA treatedmouse was switched to hMCM mRNA therapy. Interestingly, once that MCKmouse started to receive hMCM mRNA treatment, plasma MMA levels of thismouse quickly decreased from 835 μM to 153 μM within 3 days (FIG. 16A).Moreover, this mouse gained 4.8 g in body weight in just one weekfollowing crossover to hMCM mRNA therapy. After the fifth IV dose, theMCK mice were given a 10 day washout period (FIG. 16B). Interestingly,there was a partial rebound of plasma MMA to 955+/−SD μM in these mice10 days following their last hMCM mRNA injection (FIG. 16A). Moreover,during this washout period, decreased body weight was observed in thesemice (0.13+/−SD to 2.8+/−SD g) from day 6 to day 10 (FIG. 16B). When MCKmice were re-dosed with 0.2 mg/kg IV hMCM mRNA (encoding SEQ ID NO: 734)after the 10 day washout, plasma MMA levels decreased to 226+/−μM andbody weight increased from 1.38+/−g to 3.81+/−g in 4 days. These datademonstrates that LNP-formulated hMCM mRNA is efficacious in loweringplasma MMA levels and simultaneously increasing body weight in thismethylmalonic acidemia mouse model of the more severe Mut0 subtype.

Example 21. Expression of MCM in Liver of Wild-Type Mice Dosed withCodon Optimized MCM Human mRNA Compared to Endogenous MCM in NormalMouse and Human Liver

To assess MCM expression in vivo, codon optimized MCM human mRNA (SEQ IDNO: 734) was introduced into wild-type CD1 mice. CD1 mice were injectedintravenously with a single dose of codon optimized MCM mRNA (SEQ ID NO:734) at 0.2 mg/kg. The mRNA was formulated in lipid nanoparticles(Compound 18) for delivery into the mice via tail vein injection (N=3).Mice were sacrificed 24 hours after dosing, and MCM protein levels inliver lysates were determined by LC-MS/MS. As shown in FIG. 17, dosingat 0.2 mg/kg in wild-type mice resulted in abundant expression of humanMCM, higher than endogenous MCM found in human and normal mouse livers.

Example 22. MCM Expression in Liver of Mice Administered Codon OptimizedMCM mRNA

LNP encapsulates hMUT mRNA and distributes the mRNA to the liver, whereit is subsequently translated to its functional protein. The half-lifeof the mRNA and protein product are critical determinants of thepharmacokinetics of mRNA-based therapeutics. To understand the kineticsof hMUT mRNA, the LNP, and the encoded MUT protein, we performed a PKstudy in wild type CD1 mice in which mice were administered a single IVbolus of 0.5 mg/kg hMUT mRNA or vehicle control (NTFIX) mRNA. Our datashowed that 2 hours after administration, 939 ng/g LNP was detected inlivers and by 6 hours, LNP concentrations decreased by almost 98.5%(FIG. 18A). At 16 hours, LNP concentrations are not detectable in theliver suggesting that the LNP is highly degradable and is rapidlycleared in liver. hMUT mRNA was also cleared quickly as shown in FIG.18B, with a precipitous drop of hMut mRNA levels at 6 hours following asingle IV hMUT mRNA administration (FIG. 18B). Over 98% Mut mRNA wascleared 48 hours after administration. In contrast, human MUT protein israpidly expressed and showed a significantly longer half-life (FIG.18C). Human MUT protein is detectable 2 hours and peaks at 16 hoursafter hMUT IV mRNA administration (FIG. 18C). Our data suggested thatthe half-life if expressed human Mut is approximately 1.6 days. The datashow a decline in mRNA levels in the liver with levels reaching baselineby about 2 days. The sustained expression of MCM protein out to 5 daysfollowing a single administration of MCM-encoding mRNA demonstratespotential advantages in treating MMA patients with weekly dosing being apotentially acceptable dosing regimen to achieve therapeuticallyeffective doses in humans.

Example 23. Lipid Nanoparticle Quantification and Kidney, Liver andSpleen Analysis

In vivo analysis of lipid nanoparticles quantification, in situhybridization for kidney, and bDNA for spleen is assessed. Codonoptimized MCM mRNA (SEQ ID NO: 735) is administered to CD1 mice. CD1mice are injected intravenously with either control mRNA (NT-FIX) orcodon optimized MCM mRNA at 0.5 mg/kg. The mRNA was formulated in lipidnanoparticles (Compound 18) for delivery into the mice via tail veininjection. Mice (N=3/time point) are sacrificed after 2, 6, 16, 24, 48,72, 120, or 168 hours. Lipid nanoparticles are quantified at early timepoints (2, 6, and 16 hours). ISH of kidney and bDNA of liver and spleenare analyzed and compared to untreated CD1 mice.

Example 24. In Vivo Delivery of mRNA Encoding MCM

To assess methods of delivering MCM in vivo, codon optimized MCM humanmRNA, i.e., mRNA1, mRNA2, mRNA3, mRNA4, and mRNA5 (SEQ ID NOs: 775, 776,777, 734 and 778, respectively) or a vehicle control (non-translatingfactor IX, NTFIX) mRNA was introduced into wild-type CD1 mice. The micewere injected intravenously with a single dose of mRNA at 0.2 mg/kg. ThemRNA was formulated for delivery into the mice via tail vein injection(N=3 per formulation). Compositions were formulated with either MC3 orCompound 18. Mice were sacrificed 24 hours after dosing, and MCM proteinlevels in liver lysates were determined by Western blot. FIG. 19A is aWestern blot with equal loading of each lysate. FIG. 19B is aquantification of the expression patterns in the Western blot of FIG.19A. As shown in FIGS. 19A and 19B, both MC3 formulations and Compound18 formulations were effective in facilitating delivery and expressionof mRNA encoding MCM. Delivering mRNAs encoding MCM in wild-type miceresulted in expression of human MCM at levels much higher than that ofendogenous MCM in control mice.

Example 24. In Vivo MMA Level and Body Weight Effect on MCK Mice

Two MCK mice were administered weekly IV injections of 0.2 mg/kg hMCMmRNA and the other two received a vehicle control (non-translatingfactor IX, NTFIX) mRNA at 0.2 mg/kg. The study encompasses 2 doses. TheMCK mice that received hMCM mRNA injections showed decreased plasma MMAlevels 3 days following each dose (FIG. 20A). Importantly, treated MCKmice also showed significantly increased body weight which wascorrelated to decreased plasma MMA levels. See FIG. 20B. In contrast,the MCK mouse that received vehicle mRNA control (NTFIX) showed nosignificant reduction in plasma MMA levels nor increase in body weightfollowing 2 doses. One MCK mouse received the vehicle control died.

Certain exemplary mRNA sequences are shown below:

hMCM mRNATCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGA(SEQ IDAGAAATATAAGAGCCACCATGCTGCGGGCCAAGAACCAGCTGTTCCTGCTGAGCCCTCACTACCTGCGGCAGGTNO: 769)GAAGGAGAGCAGCGGCAGCCGGCTGATCCAGCAGCGGCTGCTGCACCAGCAGCAGCCCCTGCACCCCGAGTGGGCCGCCCTGGCCAAGAAGCAGCTGAAGGGCAAGAACCCCGAGGACCTGATCTGGCACACGCCCGAGGGCATCAGCATCAAGCCCCTGTACAGCAAGCGGGACACCATGGACCTGCCCGAGGAGCTGCCCGGCGTGAAGCCCTTCACCCGGGGCCCCTACCCCACCATGTACACCTTCCGGCCCTGGACCATCCGGCAGTACGCCGGCTTCAGCACCGTGGAGGAGAGCAACAAGTTCTACAAGGACAACATCAAGGCCGGCCAGCAGGGCCTGAGCGTGGCCTTCGACCTGGCCACCCACCGGGGCTACGACAGCGACAACCCACGGGTGCGGGGCGACGTGGGCATGGCCGGCGTGGCCATCGACACCGTGGAGGACACCAAGATCCTGTTCGACGGCATCCCTCTGGAGAAGATGAGCGTGAGCATGACCATGAACGGCGCCGTGATCCCCGTGCTGGCCAACTTCATCGTGACCGGCGAGGAGCAGGGCGTGCCCAAGGAGAAGCTGACCGGCACCATCCAGAACGACATCCTGAAGGAGTTCATGGTGCGGAACACCTACATCTTCCCTCCCGAGCCCAGCATGAAGATCATCGCCGACATCTTCGAGTACACCGCCAAGCACATGCCCAAGTTCAACAGCATCAGCATCAGCGGCTACCACATGCAGGAGGCCGGCGCCGACGCCATCCTGGAGCTGGCCTACACCCTGGCCGACGGCCTGGAGTACAGCCGGACCGGCCTGCAGGCCGGCCTGACCATCGACGAGTTCGCGCCCCGGCTGAGCTTCTTCTGGGGCATCGGCATGAACTTCTACATGGAGATCGCCAAGATGCGGGCCGGCCGGCGGCTGTGGGCCCACCTGATCGAGAAGATGTTCCAGCCCAAGAACAGCAAGAGCCTGCTGCTGCGGGCCCACTGCCAGACCAGCGGCTGGAGCCTGACCGAGCAGGACCCCTACAACAACATCGTGCGGACCGCCATCGAGGCCATGGCCGCCGTGTTCGGCGGCACCCAGAGCCTGCACACCAACAGCTTCGACGAGGCCCTGGGCCTGCCCACCGTGAAGAGCGCCCGGATCGCCCGGAACACCCAGATCATCATCCAGGAGGAGAGCGGCATCCCCAAGGTGGCCGACCCCTGGGGCGGCAGCTACATGATGGAGTGCCTGACCAACGACGTGTACGACGCCGCCCTGAAGCTGATCAACGAGATCGAGGAGATGGGCGGCATGGCCAAGGCCGTGGCCGAGGGCATCCCCAAGCTGCGGATCGAGGAGTGCGCCGCCCGGCGGCAGGCCCGGATCGACAGCGGCAGCGAGGTGATCGTGGGCGTGAACAAGTACCAGCTGGAGAAGGAGGACGCCGTGGAGGTGCTGGCCATCGACAACACCAGCGTGCGGAACCGGCAGATCGAGAAGCTGAAGAAGATCAAGAGCAGCCGGGACCAGGCCCTGGCCGAGCGGTGCCTGGCCGCCCTGACCGAGTGCGCCGCCAGCGGCGACGGCAACATCCTGGCCCTGGCCGTGGACGCCAGCCGGGCCCGGTGCACCGTGGGCGAGATCACCGACGCCCTGAAGAAGGTGTTCGGCGAGCACAAGGCCAACGACCGGATGGTGAGCGGCGCCTACCGGCAGGAGTTCGGCGAGAGCAAGGAGATCACCAGCGCCATCAAGCGGGTGCACAAGTTCATGGAGCGGGAGGGCCGGCGGCCCCGGCTGCTGGTGGCCAAGATGGGCCAGGACGGCCACGACCGGGGCGCCAAGGTGATCGCCACCGGCTTCGCCGACCTGGGCTTCGACGTGGACATCGGCCCACTGTTCCAGACGCCCCGGGAGGTGGCCCAGCAGGCCGTGGACGCCGACGTGCACGCCGTGGGCGTGAGCACCCTGGCCGCCGGCCACAAGACCCTGGTGCCCGAGCTGATCAAGGAGCTGAACAGCCTGGGCCGGCCCGACATCCTGGTGATGTGCGGCGGCGTGATCCCGCCCCAGGACTACGAGTTCCTGTTCGAGGTGGGCGTGAGCAACGTGTTCGGCCCCGGCACCCGGATCCCCAAGGCCGCCGTGCAGGTGCTGGACGACATCGAGAAGTGCCTGGAGAAGAAGCAGCAGAGCGTGTGATAATAGTCCATAAAGTAGGAAACACTACAGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCCGCATTATTACTCACGGTACGAGTGGTCTTTGAATAAAGTCTGAGTGGGCGGChMCM mRNATCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGA(SEQ IDAGAAATATAAGAGCCACCATGCTGCGGGCCAAGAACCAGCTGTTCCTGCTGAGCCCCCACTACCTGCGGCAGGTNO: 770)GAAGGAGAGCAGCGGCAGCCGGCTGATCCAGCAGCGCCTCCTCCACCAGCAGCAGCCCCTCCACCCCGAGTGGGCCGCCCTCGCCAAGAAGCAGCTCAAGGGCAAGAACCCCGAGGACCTCATCTGGCACACCCCCGAGGGCATCTCCATCAAGCCCCTCTACTCCAAGCGCGACACCATGGACCTCCCCGAGGAGCTCCCCGGCGTCAAGCCCTTCACCCGCGGCCCCTACCCCACCATGTACACCTTCCGCCCCTGGACCATCCGCCAGTACGCCGGCTTCTCCACCGTCGAGGAGTCCAACAAGTTCTACAAGGACAACATCAAGGCCGGCCAGCAGGGCCTCTCCGTCGCCTTCGACCTCGCCACCCACCGCGGCTACGACTCCGACAACCCCCGCGTCCGCGGCGACGTCGGCATGGCCGGCGTCGCCATCGACACCGTCGAGGACACCAAGATCCTCTTCGACGGCATCCCCCTCGAGAAGATGTCCGTCTCCATGACCATGAACGGCGCCGTCATCCCCGTCCTCGCCAACTTCATCGTCACCGGCGAGGAGCAGGGCGTCCCCAAGGAGAAGCTCACCGGCACCATCCAGAACGACATCCTCAAGGAGTTCATGGTCCGCAACACCTACATCTTCCCCCCCGAGCCCTCCATGAAGATCATCGCCGACATCTTCGAGTACACCGCCAAGCACATGCCCAAGTTCAACTCCATCTCCATCTCCGGCTACCACATGCAGGAGGCCGGCGCCGACGCCATCCTCGAGCTCGCCTACACCCTCGCCGACGGCCTCGAGTACTCCCGCACCGGCCTCCAGGCCGGCCTCACCATCGACGAGTTCGCCCCCCGCCTCTCCTTCTTCTGGGGCATCGGCATGAACTTCTACATGGAGATCGCCAAGATGCGCGCCGGCCGCCGCCTCTGGGCCCACCTCATCGAGAAGATGTTCCAGCCCAAGAACTCCAAGTCCCTCCTCCTCCGCGCCCACTGCCAGACCTCCGGCTGGTCCCTCACCGAGCAGGACCCCTACAACAACATCGTCCGCACCGCCATCGAGGCCATGGCCGCCGTCTTCGGCGGCACCCAGTCCCTCCACACCAACTCCTTCGACGAGGCCCTCGGCCTCCCCACCGTCAAGTCCGCCCGCATCGCCCGCAACACCCAGATCATCATCCAGGAGGAGTCCGGCATCCCCAAGGTCGCCGACCCCTGGGGCGGCTCCTACATGATGGAGTGCCTCACCAACGACGTCTACGACGCCGCCCTCAAGCTCATCAACGAGATCGAGGAGATGGGCGGCATGGCCAAGGCCGTCGCCGAGGGCATCCCCAAGCTCCGCATCGAGGAGTGCGCCGCCCGCCGCCAGGCCCGCATCGACTCCGGCTCCGAGGTCATCGTCGGCGTCAACAAGTACCAGCTCGAGAAGGAGGACGCCGTCGAGGTCCTCGCCATCGACAACACCTCCGTCCGCAACCGCCAGATCGAGAAGCTCAAGAAGATCAAGTCCTCCCGCGACCAGGCCCTCGCCGAGCGCTGCCTCGCCGCCCTCACCGAGTGCGCCGCCTCCGGCGACGGCAACATCCTCGCCCTCGCCGTCGACGCCTCCCGCGCCCGCTGCACCGTCGGCGAGATCACCGACGCCCTCAAGAAGGTCTTCGGCGAGCACAAGGCCAACGACCGCATGGTCTCCGGCGCCTACCGCCAGGAGTTCGGCGAGTCCAAGGAGATCACCTCCGCCATCAAGCGCGTCCACAAGTTCATGGAGCGCGAGGGCCGCCGCCCCCGCCTCCTCGTCGCCAAGATGGGCCAGGACGGCCACGACCGCGGCGCCAAGGTCATCGCCACCGGCTTCGCCGACCTCGGCTTCGACGTCGACATCGGCCCCCTCTTCCAGACCCCCCGCGAGGTCGCCCAGCAGGCCGTCGACGCCGACGTCCACGCCGTCGGCGTCTCCACCCTCGCCGCCGGCCACAAGACCCTCGTCCCCGAGCTCATCAAGGAGCTCAACTCCCTCGGCCGCCCCGACATCCTCGTCATGTGCGGCGGCGTCATCCCCCCCCAGGACTACGAGTTCCTCTTCGAGGTCGGCGTCTCCAACGTCTTCGGCCCCGGCACCCGCATCCCCAAGGCCGCCGTCCAGGTCCTCGACGACATCGAGAAGTGCCTCGAGAAGAAGCAGCAGTCCGTCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCCGCATTATTACTCACGGTACGAGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC hMCM ORFATGTTAAGAGCTAAGAATCAGCTTTTTTTACTTTCACCTCATTACCTGAGGCAGGTAAAAGAATCATCAGGCTC(SEQ IDCAGGCTCATACAGCAACGACTTCTACACCAGCAACAGCCCCTTCACCCAGAATGGGCTGCCCTGGCTAAAAAGCNO: 775)AGCTGAAAGGCAAAAACCCAGAAGACCTAATATGGCACACCCCGGAAGGGATCTCTATAAAACCCTTGTATTCCAAGAGAGATACTATGGACTTACCTGAAGAACTTCCAGGAGTGAAGCCATTCACACGTGGACCATATCCTACCATGTATACCTTTAGGCCCTGGACCATCCGCCAGTATGCTGGTTTTAGTACTGTGGAAGAAAGCAATAAGTTCTATAAGGACAACATTAAGGCTGGTCAGCAGGGATTATCAGTTGCCTTTGATCTGGCGACACATCGTGGCTATGATTCAGACAACCCTCGAGTTCGTGGTGATGTTGGAATGGCTGGAGTTGCTATTGACACTGTGGAAGATACCAAAATTCTTTTTGATGGAATTCCTTTAGAAAAAATGTCAGTTTCCATGACTATGAATGGAGCAGTTATTCCAGTTCTTGCAAATTTTATAGTAACTGGAGAAGAACAAGGTGTACCTAAAGAGAAACTTACTGGTACCATCCAAAATGATATACTAAAGGAATTTATGGTTCGAAATACATACATTTTTCCTCCAGAACCATCCATGAAAATTATTGCTGACATATTTGAATATACAGCAAAGCACATGCCAAAATTTAATTCAATTTCAATTAGTGGATACCATATGCAGGAAGCAGGGGCTGATGCCATTCTGGAGCTGGCCTATACTTTAGCAGATGGATTGGAGTACTCTAGGACTGGACTCCAGGCTGGCCTGACAATTGATGAATTTGCACCAAGGTTGTCTTTCTTCTGGGGAATTGGAATGAATTTCTATATGGAAATAGCAAAGATGAGAGCTGGTAGAAGACTCTGGGCTCACTTAATAGAGAAAATGTTTCAGCCTAAAAACTCAAAATCTCTTCTTCTAAGAGCACACTGTCAGACATCTGGATGGTCACTTACTGAGCAGGATCCCTACAATAATATTGTCCGTACTGCAATAGAAGCAATGGCAGCAGTATTTGGAGGGACTCAGTCTTTGCACACAAATTCTTTTGATGAAGCTTTGGGTTTGCCAACTGTGAAAAGTGCTCGAATTGCCAGGAACACACAAATCATCATTCAAGAAGAATCTGGGATTCCCAAAGTGGCTGATCCTTGGGGAGGTTCTTACATGATGGAATGTCTCACAAATGATGTTTATGATGCTGCTTTAAAGCTCATTAATGAAATTGAAGAAATGGGTGGAATGGCCAAAGCTGTAGCTGAGGGAATACCTAAACTTCGAATTGAAGAATGTGCTGCCCGAAGACAAGCTAGAATAGATTCTGGTTCTGAAGTAATTGTTGGAGTAAATAAGTACCAGTTGGAAAAAGAAGACGCTGTAGAAGTTCTGGCAATTGATAATACTTCAGTGCGAAACAGGCAGATTGAAAAACTTAAGAAGATCAAATCCAGCAGGGATCAAGCTTTGGCTGAACGTTGCCTTGCTGCACTAACCGAATGTGCTGCTAGCGGAGATGGAAATATCCTGGCTCTTGCAGTGGATGCATCTCGGGCAAGATGTACAGTGGGAGAAATCACAGATGCCCTGAAAAAGGTATTTGGTGAACATAAAGCGAATGATCGAATGGTGAGTGGAGCATATCGCCAGGAATTTGGAGAAAGTAAAGAGATAACATCTGCTATCAAGAGGGTTCATAAATTCATGGAACGTGAAGGTCGCAGACCTCGTCTTCTTGTAGCAAAAATGGGACAAGATGGCOATGACAGAGGAGCAAAAGTTATTGCTACAGGATTTGCTGATCTTGGTTTTGATGTGGACATAGGCCCTCTTTTCCAGACTCCTCGTGAAGTGGCCCAGCAGGCTGTGGATGCGGATGTGCATGCTGTGGGCGTAAGCACCCTCGCTGCTGGTCATAAAACCCTAGTTCCTGAACTCATCAAAGAACTTAACTCCCTTGGACGGCCAGATATTCTTGTCATGTGTGGAGGGGTGATACCACCTCAGGATTATGAATTTCTGTTTGAAGTTGGTGTTTCCAATGTATTTGGTCCTGGGACTCGAATTCCAAAGGCTGCCGTTCAGGTGCTTGATGATATTGAGAAGTGTTTGGAAAAGAAGCAGCAATCTGTA hMCM ORFATGCTGAGGGCCAAGAACCAGCTGTTTCTCCTGTCGCCCCACTACCTGAGGCAGGTGAAGGAGTCCTCCGGCAG(SEQ IDCAGGCTCATTCAGCAGAGGCTGTTGCACCAGCAGCAGCCCCTGCACCCAGAGTGGGCCGCCCTCGCCAAGAAGCNO: 776)AGCTGAAGGGGAAGAACCCCGAGGACCTGATCTGGCATACGCCCGAGGGTATCTCCATAAAACCCCTCTACAGTAAGAGGGACACCATGGACCTGCCCGAGGAACTGCCCGGCGTGAAGCCGTTCACGCGGGGCCCATACCCCACCATGTACACCTTCCGGCCGTGGACCATCAGGCAATACGCCGGCTTCAGCACCGTGGAGGAGAGCAACAAGTTCTACAAAGACAACATCAAAGCCGGTCAGCAAGGGCTGAGCGTAGCCTTCGACCTGGCCACCCACAGGGGCTACGACTCCGACAACCCCAGGGTGCGCGGCGACGTGGGCATGGCCGGCGTGGCCATCGACACCGTGGAAGACACCAAGATCCTCTTCGACGGCATCCCCCTGGAAAAGATGTCCGTGTCCATGACCATGAACGGGGCCGTTATACCGGTGCTGGCCAACTTCATAGTCACCGGCGAGGAGCAGGGGGTCCCGAAGGAGAAGTTAACCGGCACGATTCAGAACGACATCCTGAAAGAGTTCATGGTGAGGAACACCTATATCTTCCCCCCCGAGCCCTCCATGAAAATCATCGCCGACATCTTCGAGTACACCGCGAAGCACATGCCCAAGTTCAACTCCATCAGCATCTCCGGATATCACATGCAGGAAGCCGGCGCCGACGCCATCCTGGAGCTGGCCTACACCCTGGCGGACGGACTGGAGTACAGCCGCACGGGCCTGCAGGCGGGCCTGACCATAGACGAATTTGCCCCGCGGCTGAGCTTTTTCTGGGGGATCGGCATGAATTTCTACATGGAGATCGCCAAGATGCGGGCCGGCAGACGGCTGTGGGCCCATCTGATCGAAAAAATGTTCCAGCCCAAAAACAGCAAGTCCCTGCTGCTGCGGGCCCACTGCCAGACCAGCGGCTGGAGCCTGACCGAGCAGGACCCGTACAATAACATCGTGAGGACCGCCATCGAGGCCATGGCCGCCGTGTTCGGCGGGACGCAAAGCCTGCACACGAACTCCTTCGACGAGGCGCTCGGCCTGCCCACCGTGAAGTCCGCTAGGATCGCCAGGAACACACAGATCATCATCCAGGAGGAGAGCGGCATCCCCAAGGTGGCCGACCCCTGGGGCGGCTCCTACATGATGGAGTGCCTGACGAACGACGTGTACGACGCCGCCCTGAAGCTGATCAACGAGATCGAGGAGATGGGCGGCATGGCCAAGGCCGTCGCCGAGGGCATCCCCAAGCTGCGCATCGAGGAGTGCGCCGCCAGGCGCCAAGCCCGGATCGATAGCGGCAGCGAGGTGATCGTGGGGGTGAACAAGTACCAGCTGGAGAAGGAGGACGCGGTCGAGGTCCTGGCCATAGACAACACGAGCGTGCGGAACAGGCAGATCGAGAAGCTCAAGAAAATCAAGAGCAGCCGGGACCAGGCCCTGGCCGAAAGGTGCCTCGCCGCCCTCACGGAATGCGCCGCCAGCGGCGACGGCAATATCCTGGCCCTGGCGGTCGATGCCAGCCGCGCTCGGTGCACCGTGGGGGAGATCACCGATGCCCTCAAGAAAGTGTTCGGGGAGCACAAGGCCAACGACAGGATGGTGTCCGGCGCCTACAGGCAGGAGTTCGGCGAAAGCAAGGAAATCACGAGCGCCATCAAGCGGGTCCATAAGTTCATGGAGAGGGAGGGCCGGAGGCCCAGGCTGCTCGTGGCCAAAATGGGCCAGGACGGCCATGACAGGGGCGCCAAGGTGATCGCCACCGGGTTCGCCGACCTCGGCTTCGACGTGGACATCGGGCCGCTGTTCCAGACGCCGCGGGAGGTCGCCCAGCAAGCGGTGGACGCCGACGTGCACGCCGTCGGGGTGAGCACCCTCGCCGCTGGGCATAAGACCCTGGTGCCCGAGCTGATCAAAGAGCTCAACAGCCTCGGCAGGCCCGACATTCTCGTTATGTGCGGCGGCGTCATCCCGCCCCAGGACTACGAGTTCCTGTTTGAGGTCGGCGTCTCCAACGTGTTCGGCCCAGGCACCAGGATCCCCAAGGCCGCCGTGCAGGTGTTGGACGATATCGAGAAATGCCTCGAGAAAAAGCAGCAGAGCGTC hMCM ORFATGCTGCGGGCCAAGAACCAGCTGTTCCTGCTGAGCCCCCACTACCTGCGGCAGGTGAAGGAGAGCAGCGGCAG(SEQ IDCCGGCTGATCCAGCAGCGCCTCCTCCACCAGCAGCAGCCCCTCCACCCCGAGTGGGCCGCCCTCGCCAAGAAGCNO: 777)AGCTCAAGGGCAAGAACCCCGAGGACCTCATCTGGCACACCCCCGAGGGCATCTCCATCAAGCCCCTCTACTCCAAGCGCGACACCATGGACCTCCCCGAGGAGCTCCCCGGCGTCAAGCCCTTCACCCGCGGCCCCTACCCCACCATGTACACCTTCCGCCCCTGGACCATCCGCCAGTACGCCGGCTTCTCCACCGTCGAGGAGTCCAACAAGTTCTACAAGGACAACATCAAGGCCGGCCAGCAGGGCCTCTCCGTCGCCTTCGACCTCGCCACCCACCGCGGCTACGACTCCGACAACCCCCGCGTCCGCGGCGACGTCGGCATGGCCGGCGTCGCCATCGACACCGTCGAGGACACCAAGATCCTCTTCGACGGCATCCCCCTCGAGAAGATGTCCGTCTCCATGACCATGAACGGCGCCGTCATCCCCGTCCTCGCCAACTTCATCGTCACCGGCGAGGAGCAGGGCGTCCCCAAGGAGAAGCTCACCGGCACCATCCAGAACGACATCCTCAAGGAGTTCATGGTCCGCAACACCTACATCTTCCCCCCCGAGCCCTCCATGAAGATCATCGCCGACATCTTCGAGTACACCGCCAAGCACATGCCCAAGTTCAACTCCATCTCCATCTCCGGCTACCACATGCAGGAGGCCGGCGCCGACGCCATCCTCGAGCTCGCCTACACCCTCGCCGACGGCCTCGAGTACTCCCGCACCGGCCTCCAGGCCGGCCTCACCATCGACGAGTTCGCCCCCCGCCTCTCCTTCTTCTGGGGCATCGGCATGAACTTCTACATGGAGATCGCCAAGATGCGCGCCGGCCGCCGCCTCTGGGCCCACCTCATCGAGAAGATGTTCCAGCCCAAGAACTCCAAGTCCCTCCTCCTCCGCGCCCACTGCCAGACCTCCGGCTGGTCCCTCACCGAGCAGGACCCCTACAACAACATCGTCCGCACCGCCATCGAGGCCATGGCCGCCGTCTTCGGCGGCACCCAGTCCCTCCACACCAACTCCTTCGACGAGGCCCTCGGCCTCCCCACCGTCAAGTCCGCCCGCATCGCCCGCAACACCCAGATCATCATCCAGGAGGAGTCCGGCATCCCCAAGGTCGCCGACCCCTGGGGCGGCTCCTACATGATGGAGTGCCTCACCAACGACGTCTACGACGCCGCCCTCAAGCTCATCAACGAGATCGAGGAGATGGGCGGCATGGCCAAGGCCGTCGCCGAGGGCATCCCCAAGCTCCGCATCGAGGAGTGCGCCGCCCGCCGCCAGGCCCGCATCGACTCCGGCTCCGAGGTCATCGTCGGCGTCAACAAGTACCAGCTCGAGAAGGAGGACGCCGTCGAGGTCCTCGCCATCGACAACACCTCCGTCCGCAACCGCCAGATCGAGAAGCTCAAGAAGATCAAGTCCTCCCGCGACCAGGCCCTCGCCGAGCGCTGCCTCGCCGCCCTCACCGAGTGCGCCGCCTCCGGCGACGGCAACATCCTCGCCCTCGCCGTCGACGCCTCCCGCGCCCGCTGCACCGTCGGCGAGATCACCGACGCCCTCAAGAAGGTCTTCGGCGAGCACAAGGCCAACGACCGCATGGTCTCCGGCGCCTACCGCCAGGAGTTCGGCGAGTCCAAGGAGATCACCTCCGCCATCAAGCGCGTCCACAAGTTCATGGAGCGCGAGGGCCGCCGCCCCCGCCTCCTCGTCGCCAAGATGGGCCAGGACGGCCACGACCGCGGCGCCAAGGTCATCGCCACCGGCTTCGCCGACCTCGGCTTCGACGTCGACATCGGCCCCCTCTTCCAGACCCCCCGCGAGGTCGCCCAGCAGGCCGTCGACGCCGACGTCCACGCCGTCGGCGTCTCCACCCTCGCCGCCGGCCACAAGACCCTCGTCCCCGAGCTCATCAAGGAGCTCAACTCCCTCGGCCGCCCCGACATCCTCGTCATGTGCGGCGGCGTCATCCCCCCCCAGGACTACGAGTTCCTCTTCGAGGTCGGCGTCTCCAACGTCTTCGGCCCCGGCACCCGCATCCCCAAGGCCGCCGTCCAGGTCCTCGACGACATCGAGAAGTGCCTCGAGAAGAAGCAGCAGTCCGTC hMCM ORFATGCTGCGGGCCAAGAACCAGCTGTTCCTGCTGAGCCCCCACTACCTGCGGCAGGTGAAGGAGAGCAGCGGCAG(SEQ IDCCGGCTGATCCAGCAGCGCCTCCTCCACCAGCAGCAGCCCCTCCACCCCGAGTGGGCCGCCCTCGCCAAGAAGCNO: 778)AGCTCAAGGGCAAGAACCCCGAGGACCTCATCTGGCACACCCCCGAGGGCATCTCCATCAAGCCCCTCTACTCCAAGCGCGACACCATGGACCTCCCCGAGGAGCTCCCCGGCGTCAAGCCCTTCACCCGCGGCCCCTACCCCACCATGTACACCTTCCGCCCCTGGACCATCCGCCAGTACGCCGGCTTCTCCACCGTCGAGGAGTCCAACAAGTTCTACAAGGACAACATCAAGGCCGGCCAGCAGGGCCTCTCCGTCGCCTTCGACCTCGCCACCCACCGCGGCTACGACTCCGACAACCCCCGCGTCCGCGGCGACGTCGGCATGGCCGGCGTCGCCATCGACACCGTCGAGGACACCAAGATCCTCTTCGACGGCATCCCCCTCGAGAAGATGTCCGTCTCCATGACCATGAACGGCGCCGTCATCCCCGTCCTCGCCAACTTCATCGTCACCGGCGAGGAGCAGGGCGTCCCCAAGGAGAAGCTCACCGGCACCATCCAGAACGACATCCTCAAGGAGTTCATGGTCCGCAACACCTACATCTTCCCCCCCGAGCCCTCCATGAAGATCATCGCCGACATCTTCGAGTACACCGCCAAGCACATGCCCAAGTTCAACTCCATCTCCATCTCCGGCTACCACATGCAGGAGGCCGGCGCCGACGCCATCCTCGAGCTCGCCTACACCCTCGCCGACGGCCTCGAGTACTCCCGCACCGGCCTCCAGGCCGGCCTCACCATCGACGAGTTCGCCCCCCGCCTCTCCTTCTTCTGGGGCATCGGCATGAACTTCTACATGGAGATCGCCAAGATGCGCGCCGGCCGCCGCCTCTGGGCCCACCTCATCGAGAAGATGTTCCAGCCCAAGAACTCCAAGTCCCTCCTCCTCCGCGCCCACTGCCAGACCTCCGGCTGGTCCCTCACCGAGCAGGACCCCTACAACAACATCGTCCGCACCGCCATCGAGGCCATGGCCGCCGTCTTCGGCGGCACCCAGTCCCTCCACACCAACTCCTTCGACGAGGCCCTCGGCCTCCCCACCGTCAAGTCCGCCCGCATCGCCCGCAACACCCAGATCATCATCCAGGAGGAGTCCGGCATCCCCAAGGTCGCCGACCCCTGGGGCGGCTCCTACATGATGGAGTGCCTCACCAACGACGTCTACGACGCCGCCCTCAAGCTCATCAACGAGATCGAGGAGATGGGCGGCATGGCCAAGGCCGTCGCCGAGGGCATCCCCAAGCTCCGCATCGAGGAGTGCGCCGCCCGCCGCCAGGCCCGCATCGACTCCGGCTCCGAGGTCATCGTCGGCGTCAACAAGTACCAGCTCGAGAAGGAGGACGCCGTCGAGGTCCTCGCCATCGACAACACCTCCGTCCGCAACCGCCAGATCGAGAAGCTCAAGAAGATCAAGTCCTCCCGCGACCAGGCCCTCGCCGAGCGCTGCCTCGCCGCCCTCACCGAGTGCGCCGCCTCCGGCGACGGCAACATCCTCGCCCTCGCCGTCGACGCCTCCCGCGCCCGCTGCACCGTCGGCGAGATCACCGACGCCCTCAAGAAGGTCTTCGGCGAGCACAAGGCCAACGACCGCATGGTCTCCGGCGCCTACCGCCAGGAGTTCGGCGAGTCCAAGGAGATCACCTCCGCCATCAAGCGCGTCCACAAGTTCATGGAGCGCGAGGGCCGCCGCCCCCGCCTCCTCGTCGCCAAGATGGGCCAGGACGGCCACGACCGCGGCGCCAAGGTCATCGCCACCGGCTTCGCCGACCTCGGCTTCGACGTCGACATCGGCCCCCTCTTCCAGACCCCCCGCGAGGTCGCCCAGCAGGCCGTCGACGCCGACGTCCACGCCGTCGGCGTCTCCACCCTCGCCGCCGGCCACAAGACCCTCGTCCCCGAGCTCATCAAGGAGCTCAACTCCCTCGGCCGCCCCGACATCCTCGTCATGTGCGGCGGCGTCATCCCCCCCCAGGACTACGAGTTCCTCTTCGAGGTCGGCGTCTCCAACGTCTTCGGCCCCGGCACCCGCATCCCCAAGGCCGCCGTCCAGGTCCTCGACGACATCGAGAAGTGCCTCGAGAAGAAGCAGCAGTCCGTC

Other Embodiments

It is to be understood that the words that have been used are words ofdescription rather than limitation, and that changes can be made withinthe purview of the appended claims without departing from the true scopeand spirit of the disclosure in its broader aspects.

While the present disclosure has been described at some length and withsome particularity with respect to the several described embodiments, itis not intended that it should be limited to any such particulars orembodiments or any particular embodiment, but it is to be construed withreferences to the appended claims so as to provide the broadest possibleinterpretation of such claims in view of the prior art and, therefore,to effectively encompass the intended scope of the disclosure.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, section headings, the materials, methods, andexamples are illustrative only and not intended to be limiting.

1-15. (canceled)
 16. A messenger RNA (mRNA) comprising: (i) a5′-terminal cap; (ii) a 5′ untranslated region (UTR); (iii) an openreading frame (ORF) encoding the human methylmalonyl-CoA mutase (MCM)polypeptide of SEQ ID NO:213, wherein the ORF is at least 98% identicalto the nucleotide sequence of SEQ ID NO:732; (iv) a 3′ UTR; and (v) apoly-A tail.
 17. The mRNA of claim 16, wherein the ORF is at least 99%identical to the nucleotide sequence of SEQ ID NO:732.
 18. The mRNA ofclaim 16, wherein the ORF is 100% identical to the nucleotide sequenceof SEQ ID NO:732.
 19. The mRNA of claim 16, wherein the 5′ UTR comprisesthe nucleotide sequence of SEQ ID NO:215.
 20. The mRNA of claim 16,wherein the mRNA comprises the miR-142-3p binding site depicted in SEQID NO:722.
 21. The mRNA of claim 16, wherein the 5′ terminal cap isCap1.
 22. The mRNA of claim 16, wherein the poly-A tail is 100 residuesin length.
 23. The mRNA of claim 16, wherein at least 95% of uridines inthe mRNA are 5-methoxyuridines.
 24. The mRNA of claim 18, wherein the 5′UTR comprises the nucleotide sequence of SEQ ID NO:215.
 25. The mRNA ofclaim 18, wherein the mRNA comprises the miR-142-3p binding sitedepicted in SEQ ID NO:722.
 26. The mRNA of claim 18, wherein the 5′terminal cap is Cap1.
 27. The mRNA of claim 18, wherein the poly-A tailis 100 residues in length.
 28. The mRNA of claim 18, wherein at least95% of uridines in the mRNA are 5-methoxyuridines.
 29. The mRNA of claim18, wherein the 5′ UTR comprises the nucleotide sequence of SEQ IDNO:215, wherein the mRNA comprises the miR-142-3p binding site depictedin SEQ ID NO:722, wherein the 5′ terminal cap is Cap 1, wherein thepoly-A tail is 100 residues in length, and wherein at least 95% ofuridines in the mRNA are 5-methoxyuridines.
 30. A pharmaceuticalcomposition comprising the mRNA of claim 16 and a pharmaceuticallyacceptable excipient.
 31. A pharmaceutical composition comprising themRNA of claim 18 and a pharmaceutically acceptable excipient.
 32. Apharmaceutical composition comprising the mRNA of claim 29 and apharmaceutically acceptable excipient.
 33. A lipid nanoparticlecomprising the mRNA of claim
 16. 34. A lipid nanoparticle comprising themRNA of claim
 18. 35. A lipid nanoparticle comprising the mRNA of claim29.
 36. A method of treating methylmalonic acidemia in a human subjectin need thereof, the method comprising administering to the humansubject an effective amount of the mRNA of claim
 16. 37. A method oftreating methylmalonic acidemia in a human subject in need thereof, themethod comprising administering to the human subject an effective amountof the mRNA of claim
 17. 38. A method of treating methylmalonic acidemiain a human subject in need thereof, the method comprising administeringto the human subject an effective amount of the mRNA of claim
 18. 39. Amethod of treating methylmalonic acidemia in a human subject in needthereof, the method comprising administering to the human subject aneffective amount of the mRNA of claim
 29. 40. A method of treatingmethylmalonic acidemia in a human subject in need thereof, the methodcomprising administering to the human subject an effective amount of thepharmaceutical composition of claim
 30. 41. A method of treatingmethylmalonic acidemia in a human subject in need thereof, the methodcomprising administering to the human subject an effective amount of thepharmaceutical composition of claim
 31. 42. A method of treatingmethylmalonic acidemia in a human subject in need thereof, the methodcomprising administering to the human subject an effective amount of thepharmaceutical composition of claim
 32. 43. A method of treatingmethylmalonic acidemia in a human subject in need thereof, the methodcomprising administering to the human subject an effective amount of thelipid nanoparticle of claim
 33. 44. A method of treating methylmalonicacidemia in a human subject in need thereof, the method comprisingadministering to the human subject an effective amount of the lipidnanoparticle of claim
 34. 45. A method of treating methylmalonicacidemia in a human subject in need thereof, the method comprisingadministering to the human subject an effective amount of the lipidnanoparticle of claim 35.